immunohistochemistry (ihc)staining steps
DESCRIPTION
Step by step IHC stainingTRANSCRIPT
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