Thermo Scientific Pierce Antibody Immunostaining Guide Design, visualize and detect Immunostaining Primary Antibodies • Secondary Antibodies • Staining Dyes • Kits Thermo Scientific Table of Contents Pierce Antibody Immunostaining Guide Page Introduction 1-3 The Cell 4-5 Mammalian Cell Type Choices 6-8 Immunohistochemistry 9-12 Immunofluorescence 13-22 Secondary Antibodies 23-27 Primary Antibodies by Cellular Structures 28-31 by Research Areas 32-43 by Cell Signaling 44-59 by Biological Processes 60-76 Left: Detection of mouse anti-α-tubulin in an A549 cell in Telophase with Thermo Scientific DyLight Dye 550-GAM. Chromosomes (orange) at the poles become Introduction diffuse, while nuclei (blue) divide into two future cells. Immunofluorescence (IF) and immunohistochemistry (IHC) are two methods commonly used to detect proteins in a cellular context. Immunofluorescent detection of proteins can be performed on both fixed cells in culture and on paraffin or frozen tissue sections. The advantages of using IF to detect cellular proteins includes the ability to visualize the subcellular location of protein(s) of interest, assess both protein expression and post-translational modifications, and design multiplex experiments. When IF detection is extended to tissues sections (IHC), a higher level of resolution is achieved because researchers are analyzing target protein(s) in a near physiological state, making it ideal for assessing normal and disease tissues. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 1 Need Antibodies? Build a Better Antibody Introduction We have over 30,000 antibodies in 42 research areas. Use our custom services to produce antibodies you can trust. Thermo Scientific Pierce Antibodies are developed for a wide The Thermo Scientific Custom Antibody Development Service variety of application needs. Our website enables you to easily leverages our experience in making more than 18,500 antibodies search by protein target and then filter by the specific assays to peptides and recombinant proteins. Our proprietary antigen that interest you. All of our antibodies are validated in the stated design tools, including the Thermo Scientific Antigen Profiler applications and are guaranteed to perform. Software and targeted antigen display produces more robust Applications antibodies that perform better in your targeted assays. • Agglutination • Immunodiffusion When you initiate a custom antibody project with us we • Competition Assay • Immunofluorescence provide you access to our online project management tool. This • ChIP Assay • Immunohistochemistry secure account gives you easy access to project information • Cytotoxicity Assay (Frozen) and allows you to provide specific instructions for your projects. • Control • Immunohistochemistry • ELISA (Paraffin) • Electron Microscopy • Immunohistochemistry • FACS (Paraffin, Frozen) • Functional Assay • Infection • Gel Shift • Immunoprecipitation • Hemagglutination Assay • Immunoradiometric Assay • Inhibition Assay • Radioimmunoassay • Immunocytochemistry • Western Blot 2 For more information and complete antibody listings, visit www.thermoscientific.com/pierce-abs With both immunofluorescence (IF) and Panel A immunohistochemistry (IHC), a fluorophore conjugated to an antibody is usedSignal for detection detected Signal detected Introduction (see Figure 1). FluorophoresBiotin Avidin can beHRP conjugated to Fluorophore a primarySecondary antibody (direct), secondary antibody (indirect),antibody or tertiary-labeled antibodies (e.g., biotin/ Secondary antibody avidinPrimary system) for signal amplification. However, withantibody multiple amplification steps the researcher may Primary antibody observe an increaseAntigen in background, making it critical Antigen to determine the best conjugation method for each detection antibody and to include both positive and negative controls. Panel B Signal detected Signal detected Biotin Avidin HRP Fluorophore Secondary antibody Secondary antibody Primary antibody Primary antibody Antigen Antigen Figure 1. Detection of antigens in immunofluorescence (Panel A) and immunohistochemistry applications (Panel B). To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 3 Immunofluorescence and Immunohistochemistry Plasma Membrane Glucose Transporter 1 Monoclonal Antibody (SPM498) #MA1-37783 Mitochondria Introduction Mitochondrial Heat Shock Protein 70 Monoclonal Antibody (JG1) #MA3-028 Lysosome LAMP1 Polyclonal Antibody #PA1-654A Microtubules Tubulin b (TBN06(Tub2.5)) Monoclonal Antibody #MA5-117332 Centrosome g Tubulin Monoclonal Antibody (TU-30) #MA1-19421 Actin Filamin (FLM NO1(PM6/317)) Monoclonal Antibody #MA5-11705 Nucleus Phospho-c-Jun (Ser73) Polyclonal Antibody #PA5-17879 4 For more information and complete antibody listings, visit www.thermoscientific.com/pierce-abs Endosome Introduction Caveolin 1 Monoclonal Antibody (7C8) #MA3-600 Golgi Trans-Golgi Network 38 Monoclonal Antibody (2F7.1) #MA3-063 Nuclear Envelope Laminin Rabbit Polyclonal #PA5-16287 Peroxisome PXF Polyclonal Antibody #PA5-12171 Nucleolus Nucleostemin Polyclonal Antibody #PA5-11440 Nuclear Pore Laminin B2/g 1 Monoclonal Antibody (D18) #MA5-11992 Endoplasmic Reticulum PDI Monoclonal Antibody (RL90) #MA3-019 To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 5 Immunofluorescence and Immunohistochemistry Mammalian Cell Type Choices Table 1. Markers commonly used to identify stem cells and to characterize differentiated cell types. Target protein expression levels should be considered when Page citation: From Appendix E: Stem Cell Markers. In Stem Cell Information [World choosing a cell type because expression levels may vary Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2009 [cited Friday, July 09, 2010] Available at <http://stemcells. significantly between cell lines. Additionally, the cell line nih.gov/info/scireport/appendixe> should closely match the desired in vivo model. For example, researchers interested in understanding cellular mechanisms Marker Name Cell Type Significance involved in liver development and disease most commonly Blood Vessel choose a hepatic-derived cell line such as HepG2. Stem cells Fetal liver kinase-1 Endothelial Cell-surface receptor protein have become a growing trend in the lab. The advantage of using (Flk1) that identifies endothelial cell progenitor; marker of cell-cell pluripotent stem cells is their ability to differentiate into virtually contacts Introduction any cell type, which also lays the foundation to understand the Smooth muscle Smooth muscle Identifies smooth muscle cells in initial stages of development and disease. The National Cancer cell-specific myo- the wall of blood vessels Institutes has a panel of 60 markers to identify stem cells and to sin heavy chain highly characterize multiple cell lines (NCI 60 panel), which is a Vascular endothe- Smooth muscle Identifies smooth muscle cells in common starting point for choosing an appropriate cell line lial cell cadherin the wall of blood vessels (see Table 1). Bone Bone-specific Osteoblast Enzyme expressed in osteoblast; Another consideration in cell line choice is whether to use alkaline phospha- activity indicates bone formation primary or immortalized/transformed cell lines. Primary cells are tase (BAP) isolated directly from tissue and represent the closest genotype Hydroxyapatite Osteoblast Mineralized bone matrix that to the intact organism under study. Primary cells closely provides structural integrity; mimic a “normal” state of protein expression. Consequently, marker of bone formation they can maintain integrity of critical signaling pathways Osteocalcin (OC) Osteoblast Mineral-binding protein uniquely which mediate all biological processes. On the downside, synthesized by osteoblast; marker of bone formation primary cells proliferate in culture for a limited amount of time (~15-20 passages) before reaching the “Hayflick limit” or Bone Marrow and Blood “crisis.” A cell which reaches the Hayflick limit will enter into Bone morphoge- Mesenchymal stem Important for the differentiation netic protein and progenitor cells of committed mesenchymal cell either a reversible state of quiescence or an irreversible but receptor (BMPR) types from mesenchymal stem metabolically active state of senescence. Primary cells are also and progenitor cells; BMPR more difficult to transfect with exogenous DNA or siRNA. identifies early mesenchymal lineages (stem and progenitor The use of immortalized or transformed cell lines bypasses cells) the limitations of primary cells. Transformed cells arise when CD4 and CD8 White blood cell Cell-surface protein markers primary cells circumvent replicative senescence by exposure (WBC) specific for mature T lymphocyte to oncogenic insults such as overexpression of oncoproteins (WBC subtype) (Ras) introduced by viruses, loss of tumor suppressor protein CD34 Hematopoietic Cell-surface protein on bone stem cell (HSC), marrow cell, indicative of a HSC function (p53), or through DNA damaging events caused by satellite, endothelial and endothelial progenitor; CD34 UV, radiation or chemicals. Immortalized cells are generated progenitor also identifies muscle primarily through the deregulation of telomere maintenance. satellite, a muscle stem cell Typically transformed or immortalized cells are