Help needed on Crypto method

From:Brenda Begg

Hi Guys,
I stained some control slides for Cryptosporidum to determine titres for 
Primary and secondary Ab. However none of the 22 slides picked up the DAB 
stain just the H&E.
Can anyone suggest why. Here is the method I used
2-Rinse in dist H2O
3-Quench in 0.3% H2O2 in 70% methanol
4-Dist H2O
5-Citrate buffer at 75C in incubator overnight
7-Rinse in Dist H2O
8-Egg white for 10 min
9-Dist H2O
10-Skim milk for 15 min
11-Dist H2O
12-Rinse in PBS
13-5% Goat serum for 15min
14-Tap off
15-Mouse anti cryptosporidium (Titre 1:200,1000,5000,10000,20000) for 30min
16-Rinse in PBS
17-Biotinylated Goat antimouse(Titre 1:200,400,600) for 30 min
18-Rinse in PBS
19-Streptavidin/HRP 1:650
20-Rinse in PBS
21-DAB for 15 min
22-Rinse in Dist H2O and counterstain with H&E

Thanks Brenda B

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