IHC protocols

1. IMMUNOHISTOCHEMISTRY PROTOCOL
Section cutting (1-3 µm) thickness on the poly-l-lysin coated slide
Put the slide at 70°C warm hotplate for 2-3 hour before starting the
procedure
De-paraffinize the slides in xylene two time- 10 min and 5
min
Dip the slides in Alcohol (50 ml) + Xylene (50 ml)
Gradient re-hydration in ethanol : (100% ethanol, 90% ethanol, 70% ethanol, 50% ethanol, 30%
ethanol) (5 min each)

2. Antigen Retrieval: 110°C for 20 min in Decloaking Chamber in retrieval
buffer
(Low pH or high pH)
Cool at room temperature for 15 min
TBS washing twice (5 min each)
Per-oxidase blocking (3% H2O2 in methanol) for 15 minutes
Distilled water twice (5 min each)

3. Primary antibody incubation for 1 hour- 1.5 hour at room temp
(If overnight then incubate at 4°C)
TBS washing three times (5 minutes each)
Secondary antibody incubation for 30 minutes at room
temperature
TBS washing twice for 5 minutes
TBS washing twice for 5 minutes each

4. Wash the slides with TBS
Wash the slides in Distilled water
Haematoxylin for 1-5 minutes
Wash in running water
DAB chromogen (1 ml DAB buffer + 1 drop (~20-30µl) of DAB chromogen)

5. Clean with xylene
Mount the slide with DPX
Label the slides
INTERPRET THE SLIDES UNDER
MICROSCOPE
Dehydrate with 70%, 90% and 100% ethanol