Immunohistochemistry (IHC)Staining Steps

Step by step IHC staining

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