Protocol
for Immunoperoxidase staining of paraffin tissue sections
- Clean slides with 95% ethanol
- Treat with a solution containing 0.3% gelatin and
0.05% chromium poatassium sulfate
- Cut tissue sections using microtome and apply to
slides
- Deparafinize in xylene for 10 minutes. Repeat twice
for a total of three treatments
- Hydrate sections through graded alcohols
- Rinse three times with distilled water
- Incubate for 10 minutes with 0.1% H2O2
in PBS to quench endogenous
peroxidase activity
- Rinse four times with PBS
- Incubate slides for 20 minutes in 10% goat serum
in PBS (suppresses non-specific binding of IgG)
- Wash with PBS
- Incubate with primary antibody for 1 hour at room
temperature or overnight at 4oC. Optimal
antibody concentration is usually from 1-10ug/ml in
PBS-BSA
- Wash three times with PBS
- Incubate with biotin-conjugated secondary antibody
for 45 minutes. Again, optimal antibody concentration
is usually from 1-10ug/ml in PBS-BSA
- Wash three times with PBS
- Incubate with streptavadin-HRP for 15 minutes. This
step should be titrated as excess streptavadin-HRP
can lead to high background
- Wash with 0.5% triton x-100 in PBS for 30 seconds
- Incubate with fresh DAB solution for 5 minutes.
This step can be lengthened if additional sensitivity
is necessary. It is a good idea to do a quick spot
check here
- Rinse with distilled water
- Counterstain with hematoxylin
- Dehydrate with alcholos and xylene
- Mount permanent coverslip
Note: Incubations are to be at room
temperature or a 4oC humidified chamber.
Antibody concentrations here may not hold for all sources.
These concentrations should be titrated for each source.
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