Mismatch Repair (MMR) Positive

PRODUCT SPECIFICATIONS:

Tissue:  Positive staining normal colon.
Fixation: Formalin 10%, Phosphate Buffered (Part 1090).
Section/Glass: Paraffin sections cut at 4 microns on Superfrost™ Plus slides.
Quality Control Stain:  MLH1, MSH2, MSH6, PMS2 quality control stained slides included.
Reactivity: Guaranteed product specific reactivity for six months from date of receipt. Revalidate after six months to verify continued reactivity. 
Storage: 15-30°C in a light deprived and humidity controlled environment.
Intended Use: To verify histological techniques and reagent reactivity.

Before using unstained control slides, review the enclosed stained slide(s) to ensure that this tissue source is acceptable for testing needs.

APPLICATION:

Newcomer Supply Mismatch Repair (MMR) Positive Control Slides provide a single tissue source that expresses positive reactivity in each of the MMR panel of four markers; MLH1, MSH2, MSH6 and PMS2.  Mismatch Repair testing is useful in screening for colorectal carcinoma (CRC), Microsatellite Instability (MSI) and Lynch Syndrome (LS).

NEWCOMER SUPPLY VALIDATION PROCEDURE:

1. 1. Heat dry sections in oven according to your laboratory protocol.
   2. Deparaffinize sections thoroughly in three changes of xylene, 3 minutes each. Hydrate through two changes each of 100% and 95% ethyl alcohols, 10 dips each.  Wash well with distilled water.
      1. 1. See Procedure Note #1.
   3. Proceed with an epitope/antigen retrieval technique approved for use in your laboratory.
   4. Rinse in distilled water; tap off excess water.
   5. Circle sections with Pap Pen Liquid Blocker (Part 6505, 6506 or 6507) to reduce reagent usage and ensure tissue coverage.
   6. Block endogenous peroxidase with freshly made 3% Hydrogen Peroxide. Incubate for 5 minutes.
      1. 1. See Procedure Note #2.
   7. Wash slides gently in distilled water. Rinse in two changes of Tris Buffered Saline.
      1. 1. See Procedure Note #3.
   8. Tap off excess buffer; apply MLH1, MSH2, MSH6 and PMS2 primary antibodies. Apply each antibody to an individual slide and tissue section.  Incubate each at room temperature for 30 minutes.
   9. Rinse slides in two changes of buffer.
   10. Tap off excess buffer; apply Amplifier. Incubate for 10 minutes.
   11. Rinse slides in two changes of buffer.
   12. Tap off excess buffer; apply HRP Polymer. Incubate for 10 minutes.
   13. Rinse slides in two changes of buffer.
   14. Prepare required quantity of DAB substrate/chromogen.
   15. Tap off excess buffer; apply DAB. Incubate for 5 minutes.
   16. Rinse slides in two changes of buffer.
   17. Counterstain with Hematoxylin Stain, Gill I (Part 1180); 1-10 dips.
   18. Rinse slides in warm tap water to blue sections.
   19. Dehydrate in two changes each of 95% and 100% ethyl alcohol. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

RESULTS:

PROCEDURE NOTES:

1. 1. Do not allow sections to dry out at any point during procedure.
   2. Dilute Hydrogen Peroxide 30%, Aqueous (Part 1206) with distilled water to a 3% (1/10) solution prior to use.
   3. Dilute Tris Buffered Saline 0.05M, pH 7.6, 10X (Part 140304) with distilled water to a 1/10 solution prior to use for all buffer rinses.
   4. Biocare MLH1 (G168-15) is the primary antibody used.
   5. Biocare MSH2 (FE11) is the primary antibody used.
   6. Biocare MSH6 (44) is the primary antibody used.
   7. Biocare PMS2 (A16-4) is the primary antibody used.
   8. Cell Marque detection and ancillary reagents are used.
   9. If using a xylene substitute, follow manufacturer’s recommendation for deparaffinization and clearing steps.

REFERENCES:

1. 1. Biocare MLH1, MSH2, MSH6 and PMS2 Antibody datasheets.
   2. Modifications developed by Newcomer Supply Laboratory.