Stupid question: Which buffer for IF?

[Thitus]

Hi,

I am still doing 40um PFA-fixed cryosections from mouse spinal cord and the quality is rather poor (weak staining). I am permeating with 0.3% triton for 1h and my blocking serum consists of:

5% Normal Goat Serum (I'm using goat secondary ABs only)
1% BSA
0.1% Glycine
0.1% L-Lysine
0.3% Triton

in 0.1M PB, pH is 7.4

Are there any alternative blocking buffer recipes? I've come across much simpler blocking buffers like BSA containing PBS-tween and so forth.
What is the basis for your blocking buffer when you do immunofluorescence on cryosections?

Thanks a lot for the input!
Thitus

[ImmunoNYC]

I use 10% donkey serum (the host of my secondary) and between 0.1 and 0.3% Triton X 100. For IF I find the triton reduces my autofluorescence (as compared to Tween). For IF I do not use BSA.

[Thitus]

Do you use PBS, PB, TBS or a different buffer for your blocking serum?

Could I leave out the glycine and l-lysine in my blocking buffer?

[ImmunoNYC]

I use PBS. Sometimes TBS, but mainly PBS. The glycine acts to reduce autofluorescence. When I need to do this I run a separate step. However I have no experience on how it functions in a blocking buffer. All I can tell you is that my staining works well enough. Also for thicker sections you are going to want to extend your incubation times.

[alicem]

For my blocking buffer all I use is goat serum 10% (or whatever you want) made up in PBS.  The purpose is to block any non-specific binding sites and I usually incubate for 1 hour but depending on tissue thickness and whether your doing slide mounted or free floating.  This is done prior to the primary antibody.  What I use to make my primary and secondary antibodies is a mixture containing 25ml PBS, 2% goat serum (500ul), and 0.3% Tritin X100 (75ul).  I may also do my washes with this solution.  This has worked for all my IF.
Hope this helps