49026 Milmont Drive, Fremont, CA 94538 Emergo Europe, Molenstraat 15, NL-2513 BH The Hague, The Netherlands Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490, [email protected] SPECIAL STAINS Alcian Blue Cat. No. SS027-50X Doc. No. 932-SS027-50X Rev. No. C Release Date: 02-Aug-2016

Intended Use This product is intended for in vitro diagnostic use on the BioGenex Xmatrx® Automated Staining System.

Summary and Explanation Alcian Blue offers comprehensive staining for acid mucins, and stains very intenselyover a varying pH range. Strongly sulphated mucins stain below pH 1.0, weakly sulfated mucins stain at pH 2.5 and above, while sialomucins (depending on their type) stain between pH 1.5 and 3.2. Alcian Blue is a water soluble, high molecular weight, copper phthalocyanin basic dye. This Alcian Blue kit stains mucins at pH 2.5. The staining results are insoluble in water or alcohol and do not fade appreciably over time.

Principle of the Procedure The copper content produces the blue coloring. After treatment with acetic acid, both sulfated and carboxylated acid mucins and sialomucins will stain blue. Nuclear fast red counter stain the nuclei a red color.

Reagents Provided Alcian Blue (HX894-14X) 1x13ml, ready to use Nuclear Fast Red Solution (HX895-14X) 1x13ml, ready to use

Reagents Required but Not Supplied EZ-DeWax™ Solution (HK584-5K) 1x500ml concentrate Super SensitiveTM Wash Buffer, 20X (HK583-5K) 1x500ml, concentrate Mounting Medium Positive Control

Storage and Handling Store all reagents at 2-8°C. Do not use after expiration dates as indicated on the reagent labels.

Specimen Preparation Fixation plays an important role in preserving the tissue structures to be visualized using this stain. 10% neutral buffered formalin is the preferred fixative; however, Bouin’s solution may also be used. Ensure that the fixed sections are adequately embedded in paraffin. Cut tissue sections to 4-5 microns.

Precautions Not regarded as a health or environmental hazard under current legislation. This product contains no hazardous material at a reportable concentration according to U.S.29 CFR 1910.1200 and EC Directive 91/155/EC.

BioGenex Xmatrx® Automated Staining System Protocol

1. Bring reagents to room temperature. 2. Load the barcode-labeled slides for the appropriate stain into the Xmatrx® Automated Staining System slide racks. Load the RFID Tagged vials into the Xmatrx® Automated Staining System reagent racks. 3. Start Special Stains software on the instrument and check the protocol parameters against the factory default settings listed below. Change any of the parameters as required. Please note that any parameter change, once saved, becomes the default setting until changed again. Use of parameter settings other than factory default requires validation by user. 4 Select “Start Scan” and the instrument will perform the steps listed in the table (if the factory default settings have been selected).

49026 Milmont Drive, Fremont, CA 94538 Emergo Europe, Molenstraat 15, NL-2513 BH The Hague, The Netherlands Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490, [email protected]

ALCIAN BLUE PROTOCOL DEFAULT SETTING:

Reagent No.of Incubation No. of After Incubations Time Rinses

Baking 1 15 min 0

XDeWax™ 3 3 min 0

Special Stains Wash Solution 4 30 sec. 0

Alcian Blue 1 30 min 8

Nuclear Fast Red 1 5 min 8

Alcohol Wash 3 10 Sec 4

Xmount 1 10 min 0

Quality Control A positive control slide – one that will display positive staining with this stain – should be included in every run. Trouble shooting 1. Follow data sheet instructions correctly. 2. Gently invert all reagents prior to use. Expected Results Acid mucopolysaccharides stain blue; nuclei stain pink. Limitations of the procedure 1. All bacteria that contain mycolic acid may stain positive, hence it is not specific for mycobacterium, although indicative. 2. The thickness of the section may affect the intensity of the staining. Necrotic tissue may exhibit non-specific staining. 3. Use tissue, which shows positive structure or element for which it is being tested. Performance Characteristics Specificity of Special Stain Alcian Blue stains are intended for use in the study of acid mucins and mucopolysaccharides. BioGenex has conducted studies to evaluate the performance of all its Special Stains products. BioGenex Special Stains have shown reproducible and consistent results when used within a single run, between runs and between lots. The products have been determined to be stable for the periods specified on the labels either by standard real time or accelerated testing methods. BioGenex ensures product quality through 100% quality control for all products released and through surveillance programs. Bibliography 1. Theory and Practice of Histotechnology, 2nd ed.DC Sheehan, BB Hrapchak, eds., CV Mosby Co., St.Louis, MO, 1980. 2. Manual pf Histopathological and Histochemical Procedures, 16th ed. Compiled by JP Pickett, Pathology Dept., Duke Medical Center, Durham, NC, 1983. 3. Theory and Practice of Histotechnological Techniques, 4th ed., JD Bancroft & A Stevens, eds., Churchill Livingstone, New York(NY), 1996. 4. Fukunaga H et al. Am J Respirat. Grit cave Med, 166:994-997(2002)