high background in my immunohistochemistry - (Jun/17/2008 )

I'm using monoclonal anti-Hsp70 and a FITC conjugated secondary in my immunohistochemistries. the tissues are cryosectioned, %10 normal donkey serum is used to block the tissue for one hour. I used a control in which every thing was same as other slides except I incubated it overnight in a solution without primary antibody. there does not seem to be any difference between this slide and the other ones. primary is used in 1:200 concentration and the secondary is used in 1:100 concentration. In this condition even if my primary antibody is working I can not distinguish it from the background ...
How can I overcome this problem?

try 1:300 for 1hr for your secondaries, also increasing the time of the washes might help (are you using donkey cos the secondary was raised in donkey or cos its a standard?)
what is your fixative - changing that can alter results (pfa, acetone, methanol?)
what is your epitope retrieval? - a better version might increase the difference between +ve and background (triton x, proteinase k, saponin?)

dom

no the secondary is raised in goat but we use the donkey serum as a standard for all of our immunostainings. The fixative we use is %4 PFA, and I don't know about antigen retrieval I just know that we dilute the primary and secondary antibodies in a TBS buffer which has %0.25 triton X and %3 donkey serum if it's what you mean by "antigen retrieval" !
what do you recommend as the washing solution? we just use 0.1 Tris ... for how long?

oh boy. (so much for short answers)

1 - serum specific to the secondary will reduce background more than a non specific
2 - fixing tissue reduces the chance of +ve labelling - epitope retrieval combats this - try 10 min in 0.2% triton x in pbs
3 - i use pbs not tbs (not sure how much of a difference that will make - but i trust it)
4 - pfa can be a bugger for covering up certain epitopes try 50/50 acetone / methanol
5 - pbs for washes (not tris - again trust)
6 - my antibodies are in 2% BSA in pbs
i get the feeling youre working from a speedy gonzalies approach to immuno - you'll get much better results if you do one step at a time with washes in between (just enough to remove the previous soln apart of course for the astringent washes after the primary and secondary - at least five min)

have fun

dom

Perfect response to all of my questions, I love this kind of communication ... !!! I'll apply your advices, let's see what happens ... maybe I add 0.1% Triton X to the washing solution (pbs) as well, what about that ?

epitope retrieval is controlled damage - triton x is your epitope retrieval - why do more damage to your tissue if theres no apparent reason - you can add 0.05% tween 20 to your pbs to help it off the slide but thats all i'd bother with

glad i could be of help - i can now start the day with a self satisfied warm glow

good luck

dom