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problem with paraffin section - protocol of paraffin section IF (Apr/20/2010 )

The following is my protocol of paraffin section IF or IHC,the diameter of my sample is about 1cm and 5um thick. Is any one can give me some suggestions. Thanks in advance!
1.Xylene for about 1 hour until the paraffin can not be seen on the section.
2. 100% ethanol: 2 minutes
95% ethanol: 2 minutes
85% ethanol:2 minutes
70 % ethanol: 2 minutes
50 % ethanol: 2 minutes
30% ethanol: 2 minutes
all the ethanol solutions were prepared in PBS , except 100% ethanol solution.
3. heat-induced epitope retrieval
Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0)
95C for 10 minutes
4.Rinse 3 x 5min PBS.
5.Block in 5% BSA in PBS for 1 hours at room temperature.( The antigen is a membrane protein, so I do not permeate the membrane)
6. primary antibody diluted in PBS with 3%-5% BSA, overnight in 4 C.
7.Rinse 3 x 5min PBS
8.fluorophore-conjugated secondary antibody diluted in PBS with 3%-5% BSA, 1 hour at room temperature.

-tantao-

Hi,

Which protein are you trying to detect?
In which tissue?

1 hr Xylene is way too long, you may have degradation of tissue. Try Xylene 2x 5 min and make all alcohols solutions in distilled water, not PBS. After 30% ethanol rinse in tap water before retrieval.

Good luck

V.

-victorius-