Eliminating background staining for ICC - ICC (Aug/15/2005 )

I am really hoping that someone can help me, I am at my wit's end with my ICC work! For some reason all my negative control coverslips are showing background staining, in a similar pattern to that of the positive cells. I have tried blocking with serum from the same species as the secondary antibody, raising the NaCl content of rinsing buffer to 2.5%, raising the pH of rinsing buffer to 9 and also adding 0.05% tween 20 to buffer. All to no avail.

If anyone can help me it would be greatly appreciated, either with ideas on why this is happening or another possible method to overcome this.

Thanks

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Hello,

Before using your coverslips (i assume being glass coverslips), do you wash them ?

if you do, what protocol do you use ?

if you don't, you should. I've noticed that coverslips you can buy always have traces of unknown nature ; i believe these to be the cause of the type of background staining you experience.

I personnally always wash my glass coverslips either with absolute ethyl alcohol or 65% nitric acid during a few hours (or overnight), before washing them several times in demineralized water, dry them in an incubator (60°C), and sterilize them (180°C, 1h).

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