Immunohistochem for cryptosporidium

From:Brenda D Begg

Hi Guys,
I've never done immunohistochemistry before and I'm trying to set up a method for cryptosporidium.I was wondering if someone could critique the following method and give me some helpful tips.
The cryptosporidium antibody is very expensive so I don't want to waste it.

2.Rinse in distilled water for 3min
3.Boil in Citrate buffer for 10 min and cool
4.Rinse in Phsophate buffered saline(PBS)
5.5% Goat serum for 15 min
6.Tap off
7.Mouse anti-cryptosporidium for 30min (manufacturer-    Biogenesis-did not give suggested titre)1:50, 1:100, 1:1000, 1:10000
8.Rinse in PBS X 3
9.Biotinylated Goat antimouse(1:100, 1:200, 1:400)
10.Rinse in PBS x 3
11.Quench 3% H2O2 in 70% methanol for 15 min
 or quench using 0.3%H2O2 in 0.1%sodium azide in PBS)
12.Rinse PBS X 3
13.Streptavidin HRP 1:500
14.Rinse in PBS X 3
15.DAB for 15 min
16.Rinse in dist. water
17.Counterstain with Hx for 3 min
18.Blue for 3 min

Thanks Brend B


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