Customize these staining steps for your lab.

2. Step1.Deparaffinization and Rehydration 1. Xylene - 5 mins. 2. Xylene - 5 mins. 3. Absolute ethanol -3 mins. 4. Absolute ethanol - 3 mins 5. 95% ethanol - 3 mins. 6. 95% ethanol - 3 mins. Step 2 Rinse Distilled water (1 – 5 min.)

3. Step 3.Target Retrieval & 4.Rinse Total: 15 – 18 mins. plus 20 mins. cooling time = 35-38 mins) 1. High - 5 mins. 2. Medium High/Low - 5 mins. 3. Medium - 5 mins. 4. Medium - 3 mins. 5. Cooling time - 20 mins. 700-800 watts microwave oven

4. 5.Block Endogenous Peroxidase Hydrogen Peroxide 3% in TBST Hydrogen Peroxide blocking - 5 mins . (Wash buffer 1 min.) Buffer Step 6 Rinse

5. Primary Antibody Step 7.Primary Antibody 20 minutes

6. Step 8.Rinse with Buffer Buffer

7. Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute Step 8 contd

8. Envision Step 9.Envision 15 minutes

9. Step 10.Rinse in Buffer Buffer

10. Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute Step 10 contd

11. DAB 11.DAB 10 minutes

12. 12.Rinse in DW DW d

13. Wash in DW 1 Minute DW

14. Hematoxylin 13.Hematoxylin 30 sec to 1 minute

15. 14.Rinse in Tap Water

16. 15.Coverslip with Aqeous medium

17. Interpretation of Results When >10% of total number of cancer cells stained by Ab = Positive

22. Scoring ER/PR