Immunohistochemistry (IHC)Staining Steps
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The document outlines the steps for DAKO IHC staining which includes deparaffinization and rehydration of the sample, antigen retrieval, blocking of endogenous peroxidase, incubation with primary and secondary antibodies, development with DAB, counterstaining with hematoxylin, and coverslipping. The process takes approximately 1-2 hours and involves rinsing between steps in various buffers and solvents.
Immunohistochemistry (IHC)Staining Steps
- 1. DAKO IHC Staining Steps 1.Deparaffinization and Rehydration 2.Rinse with Buffer 3.Target Retrieval Cool 4. Rinse in Tap Water and DW 5.Block Endogenous Peroxidase 6.Rinse with Buffer 7.Primary Antibody 8.Rinse with Buffer 9.Envision 10.Rinse with Buffer 11.DAB 12.Rinse with DW 13.Hematoxylin 14.Rinse with TW 15.Coverslip with Aqueous Resin
- 2. 1.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. 7. Distilled water (30 seconds to 1 min.)
- 3. 3.Target Retrieval 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 H2O2 Hydrogen Peroxide blocking - 5 mins . (Wash buffer 1 min.)
- 5. Primary Antibody 7.Primary Antibody 20 minutes
- 6. 8.Rinse with Buffer Buffer
- 7. Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute
- 8. Envision 9.Envision 15 minutes
- 9. 10.Rinse in Buffer Buffer
- 10. Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute
- 11. DAB 11.DAB 10 minutes
- 12. 12.Rinse in DW DW d
- 13. Wash in DW 1 Minute
- 14. Hematoxylin 13.Hematoxylin 30 sec to 1 minute
- 15. 14.Rinse in Tap Water
- 16. 15.Coverslip with Aqeous medium