Bı∙Olegend 2012–2013

Bı∙oLegend

2012–2013

Catalog and Reference Manual

A Decade of Discovery

Table of Contents Table of Contents

General Information How to Use This Catalog and Reference Manual ...... iv Catalog Abbreviations Guide ...... v Company Profile ...... vi Ordering Information ...... viii Custom Solutions Team ...... x Technical Services ...... xi Quality and Satisfaction Guarantee ...... xi Collaborate With Us ...... xii Terms and Conditions ...... xii Product Warranties and Use Limitations ...... xiv Trademarks and Patents ...... xv Antibody Product Formats ...... xvi Frequently Asked Questions ...... xvii Technical Charts & Protocols ...... 1 Products ...... 45 Mouse Immunology ...... 45 Rat Immunology ...... 159 Human Immunology ...... 179 Cytokines, Chemokines, and Related Molecules ...... 317 Mouse Cytokines, Chemokines, and Related Molecules . . . . . 325 Rat Cytokines, Chemokines, and Related Molecules ...... 365 Human Cytokines, Chemokines, and Related Molecules . . . . . 373 Bioassay-Validated Recombinant Cytokines ...... 431 Cell Biology ...... 455 Secondary Reagents ...... 551 Isotype Controls ...... 565 Buffers, Solutions and Media Additives ...... 573 Index of Catalog Numbers ...... 579 Master Index ...... 607 BioLegend Worldwide ...... Inside Back Cover

biolegend.com • [email protected] iii How to Use this Catalog and Reference Manual

To locate products quickly and easily, this catalog is organized as follows:

Main Table of Contents: Products categorized by broad theme or species of interest (e g. ., Mouse Immunology) Sectional Tables of Contents: Products listed alphabetically by product/antigen name (e g. ., CD54 or ICAM-1) and by area of biology (e .g ., Adhesion Molecules) Note: In the Mouse, Rat, and Human Immunology sections, the first products listed are antibodies to CD antigens, then antibodies are listed alpha numerically by antigen name . Antibodies to TCRs and MHC antigens (Mouse) are listed at the ends of these sections .

Master Index: Lists products by common/antigen names, CD name, and clone name .

Index of Catalog Numbers: Locates page number via catalog number .

For example, to find an antibody specific for mouse TNF receptor, type II, you can locate it several different ways in the Master Index:

➢➢ Antigen common name TNFRII ➢➢ CD name CD120b ➢➢ Clone name TR75-89 ➢➢ Catalog number 113401

Once you have found a product of interest, the product pages contain information about both the antigen and the antibody, quality control data, and useful application notes . The small boxes on the bottom of the How to Use this Reference Use and Manual to Catalog How page describe the format of the antibody/product, the catalog number, the size, and the price, as below .

Format Biotin Catalog No . 100103 100104 Size 50 μg 500 μg Price $65 $225

Species Reactivity: Listed for clones with multi-species reactivity . Additional information can be found on Cross-Reactivity Charts on pages 28 – 31 .

If you can’t find something you are looking for, it is probably already in development! So feel free to contact us: [email protected].

For continually updated product listings, visit BioLegend on-line at biolegend.com.

iv Toll Free 1.877.BIOLEGEND (246.5343) Catalog Abbreviations Guide Catalog Abbreviations Guide

For your convenience and ease of use, here is a list of abbreviations used throughout this catalog: aa: amino acids ICFC: Intracellular Staining for Flow Cytometry AKP: Alkaline phosphatase IF: Immunofluorescence microscopy APC: Allophycocyanin Ig: Immunoglobulin Av: Avidin IHC: Immunohistochemistry BA: Bioassay IP: Immunoprecipitation BCA: Bicinchoninic acid protein assay kD: Kilodalton Block: Blocking LEAF™: Low endotoxin, azide-free (formulation) C’: Complement mediated cell depletion LPS: Lipopolysaccharide CNS: Central nervous system µg: Microgram Costim: Costimulatory or costimulation mAb: Monoclonal antibody Cy: Cyanine (dye) MHC: Major histocompatibility complex Deplete or Depletion: Cell depletion Neut: Neutralization ELISA: Enzyme-linked immunosorbent assay ng: Nanogram ELISA Capture/Detection: Capture or detection reagent for NK: Natural killer cells enzyme-linked immunosorbent assay PBMC: Peripheral blood mononuclear cells ELISPOT: Enzyme-linked immunosorbent spot assay PE (or R-PE): Phycoerythrin ELISPOT Capture/Detection: Capture or detection reagent pg: Picogram for enzyme-linked immunosorbent spot assay SAv: Streptavidin EU: Endotoxin Unit SDS-PAGE: SDS polyacrylamide gel electrophoresis FA: Functional assay TCR: T cell receptor FC: Flow cytometry TNFRSF: TNF receptor superfamily FITC: Fluorescein isothiocyanate TNFSF: TNF superfamily GPI: Glycosylphosphatidylinositol Treg: T regulatory cells HRP: Horseradish peroxidase WB: Western blotting ICC: Immunocytochemistry

biolegend.com • [email protected] v Company Profile

Gene Lay, DVM—Founder, President and CEO Co-founder of PharMingen, now BD Biosciences Nan Jiang, Ph.D.—Vice President of Operations ▪▪20+ years experience in antibody reagent industry, from product development, process development to manufacturing . Craig Monell, Ph.D.—Vice President of Sales, Marketing, and Business Development ▪▪18 years in the Biotechnology industry, including leadership roles in Marketing, Business Development, R&D, and Project Management at PharMingen/BD Biosciences, Bio- Carta, and Stratagene/Agilent Technologies John Ransom, Ph.D.—Vice President of Research and Development BioLegend develops world-class, cutting-edge antibodies and ▪▪20+ years in Biotechnology, including leadership positions reagents for biomedical research, manufactured in our state- at Advanced Molecular Design, Cytori Therapeutics, Nova-

Company Profile Company of-the-art facility in San Diego, CA . Our mission is to acceler- site, and Axiom Biotechnologies ate research and discovery by providing the highest quality Kent Johnson—Chief Financial Officer products at an outstanding value, along with superior cus- ▪▪Former CFO of PharMingen, now BD Biosciences tomer service and technical support . ▪▪19 years reagents manufacturing experience, broad ERP Our product areas include cell immunophenotyping, cyto- and M&A experience, California licensed CPA kines and chemokines, adhesion, cancer research, T regu- Xifeng Yang, M.D.—Senior Director of Cell Analysis latory cells, stem cells, innate immunity, cell-cycle analysis, ▪▪20+ years research and development experience in apoptosis, and modification-specific antibodies . All of BioLe- immunology, hematology, and cell biology gend’s reagents are supported by superior customer service and come with a quality guarantee . Our aggressive prod- Shaoquan Ji, DVM/Ph.D.—Vice President of Biomarkers uct development program, through technology licensing, and Immunoassays collaborations, and internal hybridoma development, pro- ▪▪Conducted the development and commercialization of duces strategic reagents for use in a variety of applications multiplex biomarker immunoassays at LINCO Research/ including: MILLIPORE Corp . using Luminex xMAP technology ▪▪25 years experience in the areas of nutrition, physiol- ▪▪Flow cytometry and mass cytometry ogy, protein chemistry and molecular biology ▪▪ELISA and ELISPOT ▪▪Immunoprecipitation and ChIP Nancy Huang—Senior Director of International ▪▪Western blotting Distribution ▪▪Immunofluorescence microscopy ▪▪20+ years experience in Biotech industry in the areas of ▪▪Immunohistochemistry Marketing/Sales, Distribution Management, Business ▪▪ In vitro and in vivo functional assays Development, and Human Resources, including leadership role at PharMingen/BD Biosciences . BioLegend offers the broadest selection of fluorochrome conjugates for multi-color flow cytometry . With our new Custom Solutions Team, BioLegend is well positioned to offer a wide range of custom products and services tailored to our customers’ specific needs . Incorporated in June 2002, BioLegend is located in San Diego, in the midst of one of the nation’s largest and fastest growing biotechnology clusters .

vi Toll Free 1.877.BIOLEGEND (246.5343) Worldwide Offices Zender-Reinhardt, who are able to discuss projects with cus- See Inside Back Cover for Contact Information. tomers, help with multi-color flow cytometry panels, and BioLegend strives to provide quality research reagents and assist in troubleshooting . BioLegend GmbH provides full expert technical support to the scientific community world- service sales and applications support in Germany, allowing wide . We look forward to serving your research needs and customers to get their research done efficiently and at a great becoming your long term research partner . value . Company Profile

BioLegend Japan KK, in partnership with Tomy Digital Biology Co ., Ltd ., opened in Tokyo in February 2008 . Yoshiro Namai, Managing Director, and Yoko Yano, Marketing and Business Development Manager, lead a highly experienced BioLegend UK Ltd.: In May 2010, BioLegend expanded into and trained technical staff, providing immediate access to the United Kingdom and now sells directly to UK customers BioLegend’s entire portfolio of reagents and the highest through a collaboration with Cambridge Bioscience . This level of customer service and technical support . collaboration has allowed BioLegend to extend its excellent pricing and value to UK customers . Led by Dr . Vijay Mhaiskar, BioLegend Europe, established in October 2008, provides Managing Director, the BioLegend UK team provides sales convenient ordering and quick delivery for our European cus- and technical applications support for researchers in the UK . tomers . We house significant inventory in Europe, allowing for rapid delivery of products to customers throughout Europe and other parts of the world, with minimal lag time .

BioLegend Taiwan: A new R&D facility was opened in BioLegend GmbH: In April 2010, BioLegend officially became Taiwan in 2010 . a company in Germany, headed by Detlef Reinhardt, Manag- ing Director . He is joined by experienced Scientific Applica- tion Manager, Dr . Carsten Wiethe, and Sales Manager, Claudia

biolegend.com • [email protected] vii Ordering Information

How to Order from the US and Canada Our Customer Service Department is available 7:00am to 5:00pm (PST), Monday through Friday to take and process your orders .

Ordering Information On-line: Visit biolegend.com/ordering

Phone/Fax:

Phone (Toll-Free US & Canada): 1 .877 .BioLegend (1 .877 .246 .5343) Fax: 1 .877 .455 9587.

E-mail: [email protected]

For most efficient order processing, please be sure that the following information is included on your purchase order or correspondence:

1 . Name of institution or company 2 . Account number (if available) 3 . Shipping address 4 . Billing address 5 . Catalog number, description, and correct price 6 . Quantity and size required 7 . Name and phone number of purchasing agent 8 . Name and phone number of the end user 9 . Credit Card number and expiration date or Purchase Order number 10 . BioLegend quotation number (if applicable) Shipping from BioLegend All orders are shipped F O. .B . San Diego, California . Shipping and handling charges will be prepaid and added to the invoice . Domestic orders will normally be shipped by overnight air express . BioLegend reserves the right to select the best packaging and shipping method which will ensure the stability of the product and allow efficient order tracking . Our reagents are stable when shipped at ambient temperature . Please store our products under the conditions indicated on the technical information sheet upon arrival . Domestic shipments are sent overnight Monday through Thursday . International shipments are typically sent on Fridays and Mondays . viii Toll Free 1.877.BIOLEGEND (246.5343) How to Order Internationally Customers located outside of United States may order through BioLegend distributors or through BioLegend directly . We have extensive experience in shipping to many countries directly . We will ship via our preferred courier and the shipping charge will vary depending on the weight of the shipment and the destination country . Customers will be responsible for payment of duties and taxes upon shipment arrival in their respective country . Orders from outside the USA and Canada typically require prepayment before shipping .

International Direct Orders: Information Ordering See Inside Back Cover, “BioLegend Worldwide”, for complete contact Information.

Germany: Visit biolegend.com/de and click “Kontakt”, “Ordering” for ordering instructions .

United Kingdom: Visit biolegend.com/uk and click “Contact”, “Ordering” for ordering instructions .

Japan: Visit biolegend.com/jp and click “お問い合わせ ”, “Ordering” for ordering instructions .

Italy: Visit biolegend.com/it and click “Contact”, “Ordering” for ordering instructions .

All Other Countries: Same as “How to Order from the US and Canada”, but include your Tax ID number for customs, as well as a copy of the import permit/license (if applicable) .

International Distributors: Visit biolegend.com/distributors for complete list of Worldwide Distributors .

Europe Direct Orders Serviced by BioLegend Europe: For customers located in certain countries in Europe, where BioLegend is selling direct, e g. . Germany, Italy, United Kingdom, or in countries where BioLegend does not have exclusive distributors in place, the orders can be placed with BioLegend Europe via email, telephone, or fax .

E-mail Inquiries: [email protected]

E-mail Technical Support: [email protected]

Phone/Fax numbers: Visit biolegend.com/contact

Shipping from BioLegend Europe BioLegend Europe maintains majority of the catalog items in stock at our Europe warehouse . Orders will be processed and shipped daily if received before 12:00pm . Shipping and handling charges will be prepaid, based on the actual weight of the shipment, and added to the invoice, as applicable . All orders will normally be shipped by overnight air express, unless otherwise specified . BioLegend Europe reserves the right to select the best packaging and shipping method which will ensure the stability of the product and allow efficient order tracking . Our reagents are stable when shipped at ambient temperature . Please store our products under the conditions indicated on the Product Data sheet upon arrival .

biolegend.com • [email protected] ix Custom Solutions Team

Specialized Products and Services Custom Value Tailored for Your Research As with all of our products, we provide excellent and BioLegend’s Custom Solutions Team has decades of reasonable pricing for our custom solutions . combined research and manufacturing experience Our products and services are backed by our high in developing conjugated antibodies, flow cytome- quality standards, including: try antibody cocktails, ELISAs, multiplex assays, bio- ➢➢ Pre- and post-bottling testing Custom Solutions Team Custom assays, recombinant proteins, and custom antibody production . ➢➢ Strict documentation and backup of protocols and data Custom Solutions ➢➢ Use of regularly calibrated instruments and We will collaborate with you to fully understand your equipment needs, develop a specialized custom solution, and then ➢➢ Lot-to-lot consistency verified by well-defined deliver the highest caliber products and services, along specifications with our quality guarantee . Save time and money by leveraging our expertise . ➢➢ Highly-skilled personnel with certified biohazard training ➢➢ Custom Products ➢ ▪▪Custom antibody cocktails in bulk, including cell ➢ Daily monitoring of storage refrigerators and surface and intracellular targets freezers ▪▪Lyophilized antibody cocktails Custom Flexibility ▪▪Custom antibody conjugation We understand that each customer has unique project ▪▪Multiplex immunoassays on multiple platforms requirements . That’s why we are completely flexible ▪▪ELISA and ELISPOT kits and sets with custom manufacturing, packaging, labeling, bulk sizing, batch delivery timelines, etc . We are here to ▪▪Antibody screening plates meet your needs . ▪▪Recombinant protein expression

➢➢ Custom Services Let’s get started on your custom ▪▪Biomarker assay development services solution… ▪▪Sample analysis for biomarkers Email us at [email protected] or call 858-768-5809 . Or contact your local BioLegend representative . ▪▪Custom manufacturing and packaging ▪▪Monoclonal and polyclonal antibody production ▪▪Bioassay development ▪▪Additional needs? Let’s discuss . x Toll Free 1.877.BIOLEGEND (246.5343) Technical Services Technical Guarantee and Satisfaction Services,

At BioLegend, our Technical Services team is dedicated and trained to answer your questions on our products and their related applications . All of our technical service staff are highly qualified and experienced to address your needs . We welcome any suggestions or comments to help us achieve the highest level of service .

For technical support, please call us at 1-877-273-3103 (Toll-free US & Canada) or 1-858-768-5801 (Interna- tional), or fax your inquiry to 877-455-9587, or send your email to: [email protected] . Our Technical Services Department hours are 7:00 am to 5:00 pm, Pacific Standard Time, Monday through Friday .

Quality and Satisfaction Guarantee

Our strict quality control guidelines allow us to offer a 100% satisfaction guarantee on all our products . As exceptional quality and batch-to-batch consistency are of paramount importance for all of us at BioLegend, each batch must pass stringent biochemical and biological/immunological testing requirements . If, however, any product does not perform as indicated on its Technical Data Sheet, we will either immediately replace it or credit you the original purchase price .

Please contact our Technical Services Department if you have any questions or concerns about your product . Our Technical Service Scientists are available to help with assay design, suggest relevant positive controls, and offer troubleshooting tips .

Your satisfaction is our number one priority .

biolegend.com • [email protected] xi Collaborate with Us

BioLegend is committed to providing researchers with the most comprehensive and cutting-edge high quality reagents for life science research . If you have developed reagents or technologies that you feel may be of interest to the life science research community, we would like the opportunity to collaborate with you to commercialize your inventions .

We look forward to developing a mutually beneficial collaboration with you . For more information about collaboration and our technology transfer process, please contact Craig Monell, Vice President of Sales, Marketing, and Business Development, at 858-768-5887 or [email protected] .

Terms and Conditions

Pricing Prices reflect list price and are subject to change without notice . Collaborate with Us, and Terms and Conditions and with Terms Us, and Collaborate Payment Methods and Terms We accept credit card purchases on any of our products; please have your VISA, MasterCard, American Express, or Discover Card account number and expiration date available when placing an order . Purchase order numbers are required for all billed shipments . Payment terms are net 30 days . A 1 .5% monthly late charge may be charged to accounts that are over 30 days past due . Orders from outside the USA and Canada typically require prepayment before shipping .

Limited Use Label License The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity) . The buyer cannot sell or otherwise transfer

➢➢ this product ➢➢ its components or ➢➢ materials made using this product or its components

xii Toll Free 1.877.BIOLEGEND (246.5343) Terms and Conditions (continued) Terms and Conditions

to a third party or otherwise use this product or its components or materials made using this product or its components for commercial purposes .

The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing not to transfer such materials to any third party and

➢➢ to use such transferred materials and/or information solely for research and not for Commercial Purposes .

Commercial Purposes means any activity by a party for consideration and may include, but is not limited to:

➢➢ use of the product or its components in manufacturing; ➢➢ use of the product or its components to provide a service, information, or data; ➢➢ use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or ➢➢ resale of the product or its components, whether or not such product or its components are resold for use in research

If the purchaser is not willing to accept the limitations of this limited use statement, BioLegend requires immediate return of the product with a full refund .

Order Confirmation and Duplication Written confirmation of a telephone or fax order is not required . However, if confirmation of an order is sent, it should be clearly marked “CONFIRMING ORDER – DO NOT DUPLICATE ”. Duplicated shipments due to incorrectly marked confirming orders will be subject to a Restocking Fee of 20% of the order or minimum of $25, whichever is greater, plus shipping charges .

Returns A return authorization number is required on all returns . Please contact BioLegend Customer Service Department to obtain the authorization prior to returning any item . All claims for damaged merchandise during transit must be reported immediately to the shipping carrier, and then to BioLegend . Return authorization requests must be made within 10 working days of order receipt . All claims are subject to investigation before return authorization, replacement or credit can be issued .

If BioLegend makes a shipping error, we will replace the product in error or the missing product immediately at no charge . If the customer makes an error on the order and is reported to BioLegend within 10 working days, the customer may obtain return authorization and return the product(s) for partial credit . Upon receipt of authorized returned goods, a credit will be issued, less a 20% restocking fee or minimum of $25, whichever is greater, of the product(s) returned and the original shipping and handling charge . To obtain partial credit, product(s) must be returned within 15 days of receiving return authorization .

biolegend.com • [email protected] xiii Product Warranties and Use Limitations

Limited Warranty and Liability Please review specifications of the product as published in the product details section of our website or call and request a copy from our Technical Service Department before ordering . Product details on the website generally reflect the applications and conditions for use . Products should be used in accordance with the technical information sheet shipped with the product . BioLegend reserves the right to change product specifications without prior notice . All products supplied by BioLegend are warranted, to the original purchaser only, to conform to our specifications provided with the shipment . This warranty does not extend to any product that has been modified in any way by person(s) other than BioLegend personnel or to any products that are handled contrary to the technical information sheet . Should any product fail to perform as warranted, upon notification to our Technical Service Department and investigation, BioLegend shall promptly replace it at no charge . Our obligation and your only remedy is limited to such replacement of the product or a full credit of the original purchase price of the product . BioLegend reserves the right to discontinue

Product Warranties and Use Limitations Use and Product Warranties sales of any products or to change product descriptions and formulations at any time without prior notice to purchasers .

BioLegend does not make any other warranty or representation whatsoever, whether express or implied, with respect to these products . BioLegend does not make any warranty of suitability, non-patent- infringement, merchantability or fitness for any particular research use or other purposes of any product . The entire risk as to the performance of this product is assumed by buyer . In no event shall BioLegend be liable for incidental, consequential or punitive damages .

Product Use Limitations Unless otherwise clearly indicated on product labels, BioLegend products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”

xiv Toll Free 1.877.BIOLEGEND (246.5343) Trademarks and Patents

Trademarks BioLegend®, BioLegend Logo, LEAF™, The path to legendary Costar® is a registered trademark and Stripwell™ is a trade- discovery™, TruStain FcX™, ELISA MAX™, and LEGEND MAX™ mark of Corning, Inc . are the trademarks and property of BioLegend, Inc . Cy™, including Cy3, Cy5, Cy5 .5 and Cy7, is a trademark of Amer- ABTS® is a registered trademark of Boehringer Mannheim sham Biosciences Ltd . GmbH . CyAn™ ADP and MoFlo™ are trademarks of Dako Denmark A/S . BD™, BD FACSCalibur™, BD FACSCanto™, BD FACScan™, CyTOF® is a registered trademark of DVS Sciences . BD FACSVantage™ SE, BD FACSAria™, BD LSRFortessa™, BD DyLight® is a registered trademark of Thermo Fisher Scientific Trademarks Patents and CyChrome™, BD Influx™ and BD Horizon™ are trademarks of Inc . and its subsidiaries . Becton, Dickinson and Company . FLAG® is a registered trademark of Sigma-Aldrich Co . Brilliant Violet™, Brilliant Violet 421™, BV421™, Brilliant Violet inFlux™ is a trademark of Cytopeia, Inc . 570™, and BV570™ are trademarks of Sirigen Group Ltd . Synergy™ and Eclipse™ are trademarks and Reflection® is a Cascade Blue®, Alexa Fluor®, Texas Red® are registered trade- registered trademark of iCyt Mission Technology, Inc . marks and Pacific Blue™, Pacific Orange™, and Cascade Yel- C6 Flow Cytometer® is a registered trademark of Accuri low™ are trademarks of Molecular Probes, Inc . Cytometers, Inc . Coulter® and EPICS® are registered trademarks and XL™, XL- ImageStream® is a registered trademark of Amnis Corporation . MCL™, CyAn™, Gallios™, and MoFlo™ are trademarks of Beck- man Coulter, Inc . MACSQuant™ is a trademark of Miltenyi Biotec Inc . Patents Alexa Fluor® and Pacific Blue™: U .S . Patent Nos . 5,696,157; 12G5: U .S . Patent No . 5,994,515 6,130,101; 5,798,276; 5,830,912; 6,562,632B1; 6,716,979 pending Antibodies to Human IL-4: U .S . Patent No . 5,041,381 patent applications and corresponding foreign equivalents, Antibodies to Human and Murine IL-10: U .S . Patent No . owned by Invitrogen Corp . 6,239,260 Brilliant Violet™: U .S . Patent Nos . 7,914,984; 7,811,755; Antibodies to vIL-10: U .S . Patent No . 5,756,342 7,666,594; 7,629,448; 7,214,489; 7,270,956; 7,144,950; 7,897,684 . Antibodies to Human and Murine IL-10 Receptor: U .S . Patent Licensed from Sirigen, owned by The Regents of the Univer- No . 5,863,796 sity of California . Antibodies to Human and Murine IL-17: U .S . Patent No . Allophycocyanin (APC) conjugates: U .S . Patent No . 5,714,386 6,274,711 Cyanine (Cy): U .S . Patent Nos . 4,981,977; 5,268,486 and other Antibodies to Human GM-CSF: U .S . Patent No . 5,070,013 patents pending . Antibodies to Human and Murine SLAM: U .S . Patent No . R-Phycoerythrin (R-PE) and Allophycocyanin (APC): U .S . Patent 5,576,423 Nos . 4,542,104 and 5,714,386 Antibodies to Mouse IL-6 Receptor, Mouse IL-9, Human IL-9: PE-Cy7: U .S . Patent No . 4,542,104 U .S . Patent No . 5,157,112; European Patent No . 0361284 APC-Cy7: U .S . Patent No . 5,714,386 Antibodies to Human IL-9 Receptor: U .S . Patent No . 5,962,269; Peridinin chlorophyll protein (PerCP): U .S . Patent No . 4,876,190 European Patent No . 0643719 CD44: U .S . Patent No . 5,108,904 Antibodies to Human and Murine CD317: U .S . Patent No . CD62P: U .S . Patent No . 4,783,330 7,049,408 Antibody to Human Fusin (clone 12G5): U .S . Patent No . Annexin V: U .S . Patent No . 5,834,196 5,994,515 Antibodies to T-bet: U .S . Patent Nos . 7,745,581; 7,393,944; 7,365,169 Antibodies to p27Kip1: U .S . Patent Nos . 5,688,665; 6,635,450; 6,242,575; 6,355,774 Antibodies to Human Phospho-ATM (Ser1981): U .S . Patent No . 7,108,992 Antibodies to Gamma-H2A: U .S . Patent Nos . 6,362,317; 6,884,873 Antibodies to Human KIR2DL4: U .S . Patent No . 7,435,801

biolegend.com • [email protected] xv BioLegend Antibody Formats

Format Application Format Application

Purified FC, ELISA Cap ., WB, IP, IHC, IF Tandem Dyes

LEAF™ Purified* Bioassays, ELISPOT Cap ., PE/Cy5 (Cy-Chrome™) FC Blocking/Neutralization PE/Cy7 FC Biotin FC, ICFC, ELISA Det ., ELISPOT Det ., IHC, IF PerCP/Cy5 .5 FC, ICFC

Brilliant Violet 421™ FC, ICFC, IF APC/Cy5 .5 FC

Brilliant Violet 570™ FC, ICFC APC/Cy7 FC BioLegend Formats Antibody FITC FC, ICFC, IF Applications key: FC: Flow Cytometry PE FC, ICFC ELISA Cap ./Det .: ELISA Capture/ Detection ELISPOT Cap ./Det .: ELISPOT Capture/Detection APC FC, ICFC WB: Western Blotting IP: Immunoprecipitation Alexa Fluor® 488 FC, IF ICFC: Intracellular staining for Flow Cytometry Alexa Fluor® 647 FC, IF IHC: Immunohistochemistry IF: Immunofluorescence Microscopy Alexa Fluor® 700 FC

Pacific Blue™ FC, IF

PerCP FC, ICFC

Cy3 IF

DyLight™ 405 FC, IF

DyLight™ 488 FC, IF

DyLight™ 594 FC, IF

DyLight™ 649 FC, IF

*BioLegend’s LEAF™ (Low Endotoxin, Azide-Free) purified antibodies are specifically designed for functional analyses, providing the most accurate results with minimal negative effects . LEAF™ purified antibodies contain no preservative, are 0 .2 µm filter sterilized, tested for sterility, and endotoxin level is <0 1. EU/µg of the protein (<0 .01 ng/µg of the protein) as determined by the LAL test . xvi Toll Free 1.877.BIOLEGEND (246.5343) Frequently Asked Questions

General 1 . What is the shelf life of BioLegend products? Most of our products (except some buffers or unless stated otherwise) have a guaranteed shelf life of one year from date of receipt by the end-user, under proper storage and handling conditions as instructed on our Product Data Sheets . For recombinant proteins, the minimum guaranteed shelf-life is 6 months from the date of receipt by the end-user . Frequently Asked Questions 2 . What are the concentrations of your antibodies? For antibodies offered in µg sizes, concentrations are typically (exceptions for some products) as follows:

APC, APC/Cy5 .5, APC/Cy7, PE, PE/Cy5, PE/Cy7, “Support” . If your product isn’t covered in this section, PerCP, PerCP/Cy5 .5 0 .2 mg/mL please contact BioLegend Technical Services . 6 . What is BioLegend’s Quality Policy? Purified, Biotin, FITC, Alexa Fluor® 488, Alexa Fluor® 647, Alexa Fluor® 700, Pacific Blue™ 0 .5 mg/mL 100% Satisfaction Guarantee . If BioLegend’s product does not perform as described on its product data sheet, we’ll replace it or refund 100% of the original LEAF™ Purified 1 .0 mg/mL purchase price . 7 . What is BioLegend’s Return Policy? For the antibodies offered per test, per plate, or per All claims for merchandise damaged during transit Western blot, concentrations vary, depending on opti- must be reported immediately to the shipping carrier, mal titer in the assay . If you have the Product Lot#, use then to BioLegend . our Concentration Lookup tool at biolegend.com/con- For all other returns, return authorization is required, centrationlookup or contact our Technical Services at and requests must be made within 10 working days of 877-273-3103 or [email protected] . order receipt . Obtain a return authorization number from our Customer Service department . All claims are 3 . In what formats and for what applications do you offer your antibodies? subject to investigation before return authorization, replacement, or credit can be issued . See page xvi for list of all BioLegend antibody formats and applications . If your order was placed incorrectly, the product(s) may be returned for partial credit, and the return must 4 . Multiple clones are available for a particular spec- be postmarked within 15 days of the return authorization ificity . How do I choose the right one? date . Upon receipt of authorized returned goods, a credit Check the Product Data Sheets on-line to review appli- will be issued, less a 20% restocking fee or $25, whichever cations/characteristics of each clone . If you are not is greater, and the shipping and handling charge . looking for a specific clone, we recommend using the most popular clone based on PubMed or Google 8 . Does BioLegend manufacture antibodies accord- Scholar . This will give you an idea on how often a ing to GMP guidelines? clone is used compared to another . Although BioLegend does not manufacture antibodies If you still have questions, please contact BioLegend in a certified GMP facility, our products are manufactured according to GMP guidelines . For more information, visit Technical Services at: our Quality Control page at biolegend.com/QC . Toll Free Phone (U .S ., Canada): 1-877-273-3103 Phone: 858-768-5801 Fax: 877-455-9587 9 . What is the epitope of the antibody? E-mail: [email protected] BioLegend does not epitope map antibodies . Some- 5 . How do I obtain a Material Safety Data Sheet times this information is indicated on the data sheets, (MSDS) for your products? if published, but generally, we will not know the exact epitope of the antibody . On each product data sheet there is a link to all of the available MSDS . Or check our website under

biolegend.com • [email protected] xvii 10 . How do I look for reagents that are cross- reactive with a different species (e g. . Rhesus or Cynomolgus)? See Cross-Reactivity Charts on pages 28–31 . You may also go to biolegend .com and perform an Advanced Search, which contains a drop-down menu for Species Reactivity, or visit our Non-Human Primate page at biolegend.com/nhp . Ordering 11 . What is the advantage of ordering per test, plate, or µl instead of per mg? Antibodies offered per test, plate, or µl have been pre- products are at a defined concentration regardless titrated for optimal performance in flow cytometry, of the optimal usage . Regardless of format, it is very ELISA, or Western Blot, respectively . Pre-titrated anti- important to use the correct isotype control at the bodies provide greater convenience, with savings of same amount (µg) as the antibody of interest . If the time and reagents because antibody titration is not concentration of the antibody is not on the vial, then required . use our Concentration Lookup tool at biolegend.com/ concentrationlookup or contact our Technical Services Note, a particular antibody packaged in a g format μ at 877-273-3103 or [email protected] . (untitrated) is not necessarily from the same batch number as the test format (pre-titrated) of that antibody, 16 . What is the difference between the “FC” and but both formats must pass the same QC testing crite- “ICFC” formulations? Do they have different ria . Please also note that the test format of an antibody optimal concentrations of utilization? does not always have a lower concentration than the μg FC is for cell surface staining, and ICFC is for intracellular format; this information is batch-specific . (IC) staining . Yes, the concentrations can be different . The ICFC antibodies and isotypes have optimized fluo- 12 . How are BioLegend reagents shipped? rophore conjugation properties to give optimal perfor- Most products are shipped at ambient temperature . mance for IC staining . Our products are sufficiently stable to maintain opti- Frequently Asked Questions Asked Frequently 17 . Can I use less volume or fewer cells than the mal performance after overnight shipping . recommended amount? 13 . When can I expect to receive my order if I order Yes, you can use fewer cells or less volume per test, as directly from BioLegend? long as the antibody concentration does not change . For U .S . and Canada orders placed by 3 pm PST Mon- For example, if you decrease the staining volume from Thur, if products are in-stock, they are shipped the 100 to 50 µl, you can use half the amount of antibody . same day, via Federal Express overnight, so you can But, if you decrease your cell numbers from 1 million expect to receive the products the next day . Interna- to 100,000 cells and your staining volume is still 100 µl, tional direct orders are shipped on Mondays and Fri- then you should still use the data sheet recommended days, and typically arrive within 2 – 10 days, depending amount of antibody . on how long they are in customs . 18 . How does BioLegend choose the optimal concen- 14 . Why should I order from an international tration of an antibody in flow cytometry? distributor? Generally, we test 4 to 6 dilutions with the most com- For direct orders shipped outside the U .S ., shipping monly used target cells for the titration curve, then costs typically start at USD $50 for antibody orders and determine the optimal concentration based on the $100 for dry ice shipments . This may vary country to S/N (Signal/Noise) ratio . We choose the concentration country . Additionally, prepayment is typically required before the S/N decreases, so the optimal concentration for direct international shipments, and taxes and duties is at its saturation, if possible . will be paid by the recipient upon shipment delivery . 19 . Can I use the antibody at a lower concentration In some countries, the customer needs to apply for an than the recommended amount? import permit . When ordering from a distributor, the payment terms are more flexible, the customer and Possibly; we always recommend performing a titration . technical support is more immediate, and the customer Sometimes less antibody is suitable for your experi- has greater convenience in not having to deal with cus- mental needs, particularly if antibody saturation in toms agencies, duties/taxes, and currency exchange . your staining is not a concern . The % positive staining should be maintained at your selected concentration; i .e ., if while you are titrating, the population % drops, Flow Cytometry then you are not staining all the cells and should use a 15 . What is the difference between test and µg sizes? higher concentration . The test size products are pre-titrated for optimal staining of 1 million cells in 100 µl volume . The µg size xviii Toll Free 1.877.BIOLEGEND (246.5343) 20 . Can I use BioLegend antibodies for cell sorting? 25 . What is the most common reason for lack of IL-17 Yes, in general, our antibodies can be used for cell staining? sorting, but we do not routinely test this in-house . Most failures of IL-17 I/C staining are due to a stimula- Please note, the fluorophore conjugates are not endo- tion problem . PMA/Ionomycin in liquid form is very toxin tested and contain 0 .09% azide . Typically, this low unstable . We recommend storing single use aliquots amount of azide will not affect viability of the cells . As at -20 degrees for no longer than 6 months . The ali- for activation of cells, typically, the incubation time with quots should be thawed at ambient temperature, and the antibodies for staining is short (~20 minutes), and diluted in PBS to a working concentration immediately this should not cause activation . Incubation of anti- before stimulation . bodies with cells at 4°C or on ice will help prevent activation . Brilliant 21 . What is the F/P ratio for my fluorophore- Violet™ conjugated antibody? 26 . What is Brilliant All antibodies labeled with PE, APC and their tandem Violet™? Frequently Asked Questions dyes have a F/P of 1:1 . Other formats, such as FITC or Alexa dyes, have various F/P ratios, typically within the Brilliant Violet™ is range of 3 – 7 . family of fluorescent polymers created 22 . Will the 0 .09% sodium azide in my fluorophore- by Sirigen based conjugated antibody affect the viability of my on Nobel Prize win- flow sorted cells? ning chemistry . The Diluting the antibodies for staining also dilutes the first in the series is sodium azide to a concentration likely not to be toxic Brilliant Violet 421™, to the cells . Since the cells are left in contact with excited by the violet the antibody (and the azide) for only 1/2 hour, then laser at 405 nm and washed, there will be little or no residual azide in the emitting optimally sorting buffer, so the chances of residual toxicity are at 421 nm, which very low (per the Purdue Cytometry website) . can be used in place of Pacific Blue™, 23 . Which FOXP3 clone should I use? Horizon™ V450, eFluor® 450, and Alexa Fluor® 405 . Bril- For detecting human FOXP3, based on our internal liant Violet 570™ is also excited by the violet laser, testing data, both 259D and 206D clones give good emitting optimally at 570 nm, and can be used in place staining . Please see the Treg homepage for the dif- of Pacific Orange™, depending on the filter used . ferences between clone 206D or 259D . 206D recog- 27 . How does Brilliant Violet™ perform compared to nizes human FOXP3 epitope in the region of amino other common fluorophores? acids 105 – 235 . The 259D antibody recognizes human FOXP3 epitope in the region of amino acids 105 – 235 . Brilliant Violet 421™ antibody conjugates give an exceptional signal-to-noise ratio, in some cases greater In the following publication the authors state 206D than 10-fold better than Pacific Blue™ . It gives PE- clone is superior to FOXP3 antibody clones 236A/E7 and equivalent brightness or better on the violet laser . Bril- PCH101 . However, they did not compare with clone 259D . liant Violet 570™ antibody conjugates also provide Grant, J ., et al. Cytometry B Clin Cytom. 2009 Mar; much improvement over Pacific Orange™, by as much 76(2):69-78 . as 6-fold . Both of the antibodies perform well in our hands and 28 . Can Brilliant Violet™ be used for microscopy? we do not have a particular preference . However, in the Yes, Brilliant Violet 421™ is exceptionally bright and current literature, scientists seem to prefer 206D . For photostable, making it an excellent choice for micros- other mammalian species, 150D is recommended . copy . Brilliant Violet 570™ has not been fully validated for microscopy . 24 . Can FOXP3 be co-stained with IFN-γ and surface markers? 29 . How can I be sure Brilliant Violet™ will work for me? We have tested IFN-γ with FOXP3 staining and it does work . It is recommended to use BioLegend’s FOXP3 Select Brilliant Violet™ antibodies have been beta- Fix/Perm Buffer set and the FOXP3 staining protocol . tested and verified by over 20 premiere immunology The only addition is to add the cytokine antibody at research labs world-wide, in a variety of applications the same time as the FOXP3 antibody . In order to cap- including multi-color flow cytometry, microscopy, tet- ture cytokines intracellularly, cells should be stimu- ramer staining, and cell sorting, as well as a variety of lated in the presence of monensin or brefeldin A . The instruments, including common flow cytometers and Intracellular Staining Guide on page ### is a useful ref- the Amnis ImageStream . BioLegend QC tests every lot erence for generating various cytokine producing tar- of product pre- and post-bottling . As with all prod- get cells . ucts, we back every Brilliant Violet™ product with our 100% guarantee .

biolegend.com • [email protected] xix 30 . What bandpass filter should I use to detect 37 . How do I know how much isotype control to use Brilliant Violet 421™ and Brilliant Violet 570™? for test size products? We recommend using the standard 450/50 filter to Match your isotype to the concentration of the primary detect Brilliant Violet 421™, which is the same filter antibody . If your vial label does not contain the anti- used to detect Pacific Blue™ . For Brilliant Violet 570™, body concentration, call or email tech support and ask we recommend the 585/42 bandpass filter, commonly for the concentration of your lot of antibody . If you used for Pacific Orange™ . Other filters can be utilized, have the Product Lot#, use our Concentration Lookup depending on the panel of fluorophores used . See our tool at biolegend.com/concentrationlookup or contact on-line Fluorescence Spectra Analyzer for complete our Technical Services at 877-273-3103 or techserv@ excitation and emission data . biolegend.com . 31 . Will Pacific Blue™ antibodies be discontinued? Most Pacific Blue™ antibodies will not be discontin- ELISA ued, and we will continue to make them available to 38 . What is the difference between LEGEND MAX™, customers . As more customers transition to Brilliant ELISA MAX™ Deluxe, and ELISA MAX™ Standard? Violet™, discontinuation will be based upon customer LEGEND MAX™ Kits contain Pre-coated Plates and all demand for products . necessary reagents, ready to use . Most convenient, shortest protocol, full analytical and biological valida- Functional Antibodies tion . We recommend LEGEND MAX™ Kits for users who 32 . I am going to do a functional assay . Which grade are new to ELISAs or are short on time . of purified antibody should I choose? ELISA MAX™ Deluxe Sets contain Uncoated Plates, BioLegend’s LEAF™ (Low Endotoxin, Azide-Free) purified Pre-titrated Capture and Detection Antibodies, Avidin- antibodies are specifically designed for functional anal- HRP, Recombinant Standard, Coating Buffer, Assay Dilu- yses, providing the most accurate results with minimal negative effects . LEAF™ purified antibodies are tested for sterility, and endotoxin is <0 .01 ng/µg of antibody . 33 . Can I get a discount for a bulk antibody purchase? Yes, we do provide discounting based on quantity of antibody requested . Please contact us at sales@biole- Frequently Asked Questions Asked Frequently gend.com to obtain a quote . 34 . Does BioLegend test each LEAF™ antibody by functional assay? No, BioLegend does not test LEAF™ antibodies by functional assay . Due to the complexities and varying uses of biofunctional antibodies, and because of the large product portfolio, BioLegend does not currently perform functional assays as routine antibody QC . However, we do provide references in which the anti- ent, and TMB Substrate Reagent . Convenient and cost- bodies were used for functional assays, and we per- effective option . form QC to verify the specificity and quality based on ELISA MAX™ Standard Sets contain Pre-titrated Cap- our strict specifications . ture and Detection Antibodies, Recombinant Stan- 35 . Does BioLegend test each LEAF™ antibody for dard, and Avidin-HRP . Most cost-effective, plates not potential pathogens? included, optimization required . No, BioLegend does not test for pathogens in-house . 39 . Can I mix reagents from different ELISA MAX™ However, we can recommend an outside vendor to Sets, or use them for other applications? perform this testing as needed . This is not recommended . The ELISA MAX™ reagents are optimized for a particular set lot, and they are not Isotype Controls recommended for applications other than ELISA . 36 . How do I choose the right isotype control for the 40 . How should the plates be stored after coating? primary antibody I’m using? If coated plates cannot be used immediately, they Refer to the listed isotype and format of each primary should be sealed and stored overnight at 4°C . antibody, and match the isotype control appropriately . For example, if you have a PE conjugated primary anti- 41 . What kind of coating buffer should I use for my body with Rat IgG1, k isotype, use PE Rat IgG1, k isotype ELISA? control Catalog # 400407 or 400408 as your isotype This may be dependent on the cytokine being control . For your convenience, most products have their detected, but in general, scientists use PBS, pH 7 .4 or isotype control listed under their “related products” on carbonate buffer at pH 9 .5 . our website . xx Toll Free 1.877.BIOLEGEND (246.5343) ▪▪If possible, read the plate at 570 – 590 nm for background subtraction . ▪▪Improve reproducibility by controlling pipetting error or washing with a larger volume of wash buffer . ▪▪If available with your software, use 5-PL or 4-PL curve-fitting (instead of linear curve-fitting) for better calculation at the lower end of the curve . 47 . Should serum or plasma samples be used in my ELISA experiment? This depends on the targets being detected and the biological processes . Customers are advised to study the difference between serum and plasma for the tar-

42 . Why do you recommend not to use sodium azide gets of interest and decide on the sample type to be Frequently Asked Questions in ELISA buffers? used for quantification . Depending on target, there may be a difference in concentration between serum Azide is an irreversible inhibitor of horseradish peroxi- and plasma . In general, plasma/serum samples should dase (HRP) . be free of particulate matter, contain no excess lip- 43 . Is there a time frame in which the coated ELISA ids, and have no hemolysis . These types of contami- plates should be used? nants will contribute to background and adversely affect the precision of the assay . To ensure precise The plates can be coated with the capture antibody measurements, the most important factor is consis- and stored sealed at 4°C for 2-3 days . During the tency in preparation of plasma or serum samples . That blocking step, the plate can be stored in blocking buf- is, centrifuge samples at the same speed, for the same fer for 1 – 2 days at 4°C (sealed), then continue with amount of time, remove the serum or plasma immedi- rest of the protocol . The plates should not be allowed ately after centrifugation, and aliquot and freeze the to dry . samples for the same amount of time . Avoid repeated 44 . Where can I find a troubleshooting guide for freeze-thaw cycles . ELISA? 48 . Why are my samples’ cytokine concentrations biolegend.com/elisa_troubleshooting different when tested in LEGEND MAX™ Kits vs . 45 . Can I use components from different companies other ELISA Kits? for my ELISA? Cytokine kits from different manufacturers do quanti- No, this is not recommended . Different kits utilize dif- tate samples differently at the pg/mL level . The immu- ferent antibodies, and antibody specificity partially noreactivity of reagents vary among different manu- determines how much signal is detected . The recom- facturers, and for some cytokines, concentrations can binant standards in different kits also vary; some are vary a few-fold . expressed and purified by different methods . Even When quantifying cytokines at the picogram level, expressed from the same source (e g. ., E. coli), recom- the general consensus is that it is more important to binant proteins can show greater than 10-fold differ- have matching biological trends than matching abso- ences in immunoreactivity from lot to lot, primarily lute concentrations . So the same cytokine measured in due to refolding inconsistency . Different kit standards different vendors’ kits should show the same pattern of may also be produced and calibrated against different references . There is currently no universally accepted biological changes predicted for the relevant biologi- standardization for cytokine immunoreactivities . cal treatment . It is critical for kits produced by the same Each BioLegend ELISA product was developed and val- vendor to keep its assay products consistent from lot idated with reagent concentrations and protocols opti- to lot over time . Our scientists tested ELISA standards mized for best analytical robustness . Any changes to from competitors’ kits, and results showed clear dis- the reagents (standards, antibodies, matching matri- crepancies in the relative potency of the standards in ces) or protocol will affect the final assay performance . terms of their immunoreactivity . 46 . How can I obtain better ELISA signal and sensitivity? Western Blot ▪▪Increase incubation times (1st incubation, detection, 49 . What are possible causes of High Background in avidin-HRP or TMB substrate) my Western Blot? ▪▪Shake plates during incubation steps ▪▪Transfer buffers may have become contaminated . ▪▪Make sure that the standard is completely reconsti- ▪▪Post-antibody washes may not have been per- tuted before use . formed with sufficient time or volume . ▪▪Increased washing and soaking in between wash- ▪▪Blocking and incubation agents were not freshly ings to further decrease background . prepared or were too dilute .

biolegend.com • [email protected] xxi 50 . What are possible causes of No Signal/Poor Signal 55 . Why do I need to use Annexin V Binding Buffer? in my Western Blot? Annexin V binding requires the presence of calcium ▪▪Transfer efficiency may have been poor . Check pro- in the solution . BioLegend’s Annexin V Binding Buf- tein transfer by staining the gel and/or membrane . fer (Cat # 422201) is optimized for best performance of ▪▪Incorrect storage of antibodies or ECL western blot- Annexin V staining . ting detection reagents . 56 . How long can I store an antibody after I have ▪▪Insufficient protein may have been loaded on the diluted it? gel . Depending on the location of the target pro- We do not recommend extended storage after dilu- tein, membrane or nuclear preparations may be tion . Buffers without preservative may be susceptible required (instead of whole cell lysates) . to changes in pH . It is best to prepare the dilution as needed on the day of use . Long-term storage greater ▪▪Film exposure time may have been too short . than a day or two is not recommended . IHC/IF 57 . What is the concentration of the 1000X Monensin Solution (Cat #420701)? 51 . Can antibody X be used for immunohistochemistry? What concentration do I use? BioLegend’s 1000X Monensin is provided at 2 mM . Typical concentrations of monoclonal antibodies for 58 . What is the concentration of the 1000X Brefeldin use in IHC are from 5 – 25 µg/mL . While we do not test A Solution (Cat #420601)? for the IHC application in-house, if an antibody has BioLegend’s 1000X Brefeldin A is provided at 5 mg/mL . been published for use in this applica- 59 . The Brefeldin A solution freezes at 4°C . Is the solution, we indicate this tion still OK? on the datasheet . Yes . We recommend thawing the solution in warm In addition, you can water after removing from the refrigerator . The do a lit search with Brefeldin A is dissolved in DMSO, which freezes at 18°C, the clone name and so this is expected . No problems after freeze-thaw immunohistochem- cycles have been reported to us . If still concerned, the istry/paraffin/frozen Brefeldin A can be aliquotted to minimize freeze-thaw to check the protocol cycles . Frequently Asked Questions Asked Frequently details . 52 . Do I need to per- 60 . How do I choose between Monensin or Brefeldin form antigen A solution? retrieval on my We recommend optimization for each cell type and formalin-fixed, protocol . Generally, for shorter stimulations (6 hrs or paraffin-embed- less), use Brefeldin A . Monensin is recommended for ded samples prior longer incubations, as Brefeldin A is more toxic for lon- to staining? ger stimulations . In most cases, this is true . Antigen retrieval helps 61 . When do I use FluoroFix™? both the accessibility of the antibody to the tissue and FluoroFix™ (cat# 422101) is designed for fixation of cells counteracts the fixation effects on the recognized epi- stained with fluorescent labeled antibodies, including topes . BioLegend does not test antibodies for IHC, so tandem dyes . check the application references for use of the antibody in paraffin-embedded samples . 53 . Can I use GFP-expressing cells with Alexa 488 or FITC? No, due to spectral emission overlap, you will not be able to distinguish between GFP and AF488/FITC . Buffers & Solutions 54 . Why do I need to use BioLegend’s FOXP3 Fix/ Perm Buffer Set? BioLegend’s FOXP3 antibodies are optimized for use with our FOXP3 Fix/Perm Buffer Set (Cat # 421403) . Buffer sets from other vendors may not work ade- quately with our antibodies . For this reason, we highly recommend using our FOXP3 Fix/Perm Buffer Set .

xxii Toll Free 1.877.BIOLEGEND (246.5343)