Immunofluorescence advice needed!

[Simon Twigger]

Hi there, Im just starting some immunocytochemistry, nothing too fancy,
just growing tissue culture cells on coverslips, fixing them using
alcohol/ether and then immunostaining. Im using fluorescent secondary
Ab and visualising them using the usual filters.Im having a problem with my
secondary antisera giving low to moderate levels of non-specific binding. 
I am using anti-rabbit-FITC and boehringer's anti-DIG-rhodamine. Im using
them at a final concentration of 2ug/ml in a blocking solution containing
50mM tris pH7.6, 150mM NaCl and BSA (25mg/ml). Both secondary Ab
give similar levels of binding in the absence of primary antisera.

The procedure I am using is:

fix using EtOH/Ether.
block 45m using blocking solution mentioned above
primary Ab (10ug/ml in blocking soln)
wash 3x TBS
secondary Ab (2ug/ml in blocking soln)
wash 3x TBS
mount coverslip on slide.


Is it normal to get low levels of non-specific binding with secondary Ab
alone, or should I get no signal whatsoever if I have no primary Ab?

When I use the primary Ab some cells come up very strongly, the majority
are moderately stained, and the remainder are stained to a lesser degree.
My protein should be expressed in all these cells, so i would expect a
fairly uniform level of staining in all the cells. Does this mean my
immunostaining technique is not working as well as it should, leading to
only a few cells being successfully labelled, or is this a real result?!


Can anyone recommend a good handbook covering these techniques that I can
refer to for general hints and troubleshooting?

Any advice/comments anyone can give me would be most welcome.
Thanks in advance, 


	Simon..

Dr Simon Twigger,
Dept. Biochemistry,
Medical College of Wisconsin, Milwaukee. WI.