Immunoperoxidase Protocol for Cultured Cells (
1.Fix the cell suspension with cooled acetone (5X106 cells/ml) for 10 mins.
2.Wash in PBS three times for 5 mins each.
3.Dry 25-100ul
of the fixed suspension on each silanized slide at room temperature (RT).
4.Incubate sections with 1% H2O2 for 10 mins at RT to
inhibit endogenous peroxidase activity. (1% H2O2: 20ul H2O2 plus 2ml PBS).
5.Wash with PBS twice for 5 mins each.
6.Incubate sections in 1.5% blocking serum in PBS for 1 hour.
(1.5% blocking serum: 30ul serum plus 2 ml PBS)
Blot excess blocking serum. Don not wash!
7.Incubate sections with primary antibody solution for 30 mins at RT, or overnight
at 4ºC freezer.
(#1 antibody dilution: Dilute the antibody with 1.5% blocking serum PBS solution, with final concentration 0.5-5ug/ml.
8.Wash with PBS three times for 5mins each.
9.Incubate sections with biotinalaed second
antibody solution, for 30 mins at RT.
(#2 antibody preparation: 5ul #2 antibody plus 15ul blocking serum, 1ml PBS)
10.Prepare
for AB enzyme reagent for step 12:
50ul A , 50ul B, mix with 2.5ml PBS, stand for 30 mins
11.
12.Incubate sections with AB enzyme reagent for 30 mins at RT.
13.
14.Incubate sectionswith peroxidase substract for 30 seconds-10 mins. Observe the color developing process under microscopy.
(Peroxidase substrate: 125ul
10X substrate buffer,25ul 50X DAB,25ul 50X peroxidase substrate,0.8ml ddH2O).
15.Wash the sections with ddH2O for 5 mins.
16.Counterstain
sections in Gill’s formulation #3 hematoxylin for 5-10 seconds.
Immediately wash with several changes of ddH2O.
17.Dehydrate :
twice 95% ethanol for 10 seconds each, twice 100% ethanol for 10 seconds each. Three times xylene for 10 seconds each. Wipe off excess
xylene.
18.Immediately add 1-2 drops of permounting medium and cover with a glass cover glass.