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

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.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

5.Block Endogenous Peroxidase

Hydrogen Peroxide 3% in TBST

H2O2

Hydrogen Peroxide blocking - 5 mins.

(Wash buffer 1 min.)

Primary Antibody

7.Primary Antibody

20 minutes

8.Rinse with Buffer

Buffer

Buffer BufferBuffer

Wash in Buffer x 3

Dip 10 times Dip 10 times 1 minute

Envision

9.Envision

15 minutes

10.Rinse in Buffer

Buffer

Buffer BufferBuffer

Wash in Buffer x 3

Dip 10 times Dip 10 times 1 minute

DAB

11.DAB

10 minutes

12.Rinse in DW

DW

d

Wash in DW

1 Minute

Hematoxylin

13.Hematoxylin

30 sec to 1 minute

14.Rinse in Tap Water

15.Coverslip with Aqeous medium