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 Safranin O Stain Kit Cat. No. SS040-50X
Doc. No. 932-SS040-50X 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
Safranin O staining is used for the detection of cartilage, mucin, and mast cell granules on formalin-fixed paraffin-embedded and frozen tissue sections. In this staining cartilage and mucin stain orange to red, nuclei stain black and the background is stained grey green. Safranin 'O' is used to demonstrate any changes that occur in articular disease.
Principles of the Procedure
Safranin-O is a cationic stain (basic dye) that stains acidic proteoglycan present in cartilage tissues. Safranin-O used in articular cartilage staining binds to glucosaminoglucan and shows an orange-red color. Fast green is a sulfate-group containing acidic substrate, which binds strongly to the amino group on protein and strongly stains the noncollagen sites. The intensity of safranin 'O' staining is directly proportional to the proteoglycan content in cartilage tissue.
Reagents Provided Weigert’s Iron Hematoxylin A (HX912-7X) 1x 7 ml Weigert’s Iron Hematoxylin B (HX913-7X) 1 x 7 ml Fast Green Solution (HX958-14X) 1 x 13 ml, ready to use Acetic Acid (HX917-14X) 1 x 13 ml, ready to use Safranin O (HX935-14X) 1 x 13 ml, ready to use Empty RFID vial for Weigert's Iron Hematoxylin working solution (HX912)
Reagents Required but Not Supplied
EZ-DeWax™ Solution (HK584-5K) 1 x 500 ml, concentrate Super Sensitive TM Wash Buffer, 20X (HK583-5K) 1 x 500 ml, concentrate Mounting Medium Positive Control Tissue
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. Ensure that the fixed sections are adequately embedded in paraffin. Cut tissue sections to 4-5 microns.
Preparation of working Solution Weigert's Working Solution: Add Weigert's Iron Hematoxylin Solution B to Weigert's Iron Hematoxylin Solution A in 1:1 ratio in the Empty RFID Vial for Weigert's Iron Hematoxylin working solution (HX912). Mix in order indicated, do not pour back and forth. Stable for 7 days when stored at 2-8°C. Bring to room temperature before use.
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]
Xmatrx BioGenex 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 the user. 4. Select “Start Scan” and the instrument will perform the steps listed in the table (if the factory default settings have been selected). Safranin O staining Protocol Default Setting:
Reagent No. of Incubations Incubation Time No. of After Rinses Baking 1 20 mi n 0 EZ -DeWax™ 3 3 min 0 Special Stains Wash 3 30 sec 0 Solution Weigert’s iron Haematoxylin 1 10 min 4 Fast Green 1 10 min 0 Acetic Acid 1 10 sec 0 Safranin O 1 10 min 3* Xmount 1 10 min 0 *Alcohol Wash
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 Nuclei stain black, Cytoplasm stains bluish green, Cartilage, mucin & mast cell granules stain orange to red
Limitations of the procedure 1. The thickness of the section may affect the intensity of the staining. Necrotic tissue may exhibit non-specific staining. 2. Use tissue, which shows positive structure or element for which it is being tested. 3. All tissues need to be dewaxed.
Performance Characteristics Specificity of Special Stain Safranin O is specific for acidic proteoglycan present in cartilage tissue sections.
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 Histotechnological Techniques, 4 th ed. JD Bancroft & A Stevens, eds., Churchill Livingstone, New York (NY), 301-302, 1996. 2. Carson FL: Histotechnology: A self Instructional Text, ASCP Press, Chicago (IL), 194-196, 1990.
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]