Hi Mario:
\
    Just because the tissue can be processed whole does not mean that 
antibody molecules, which are huge, will penetrate all the way to 
interesting places. The various permeablization methods make cell 
membranes more permeable by removing lipids. Do put some TritonX (try 
0.1 to 0.3%) in your primary and secondary Ab solutions.

Also
1. cut the "brain" in half or quarters to improve penetration, or cut 
Vibratome or frozen sections.
2. are you sure your Ab gives good results with buffered 
paraformaldehyde fixation for whatever time you are fixing?
3. have you tried an acid -alcohol fixative (which works differently 
than formlin)?
4. a freeze-thaw cycle, after appropriate cryoprotection, also improves 
penetration by punching holes in cell membranes.

    As far as which is the best method for your tissue+fixation+antibody 
combination, you should do a literature search (PubMed) to see what has 
been done and what their results look like.

Geoff

Mario Muscedere wrote:

> Hello,
>     I'm relatively new to immunohistochemistry, and am trying to 
> optimize a protocol for staining whole ant brains.  The brains are 
> relatively small pieces of tissue (about 300 microns x 300 microns) 
> that should be (if I understand correctly) well within the size range 
> of tissue pieces that can be successfully processed whole.  I have 
> been using the standard 4% paraformaldehyde in PBS at 4 C fix, and 
> have been incubating in primary antibody 1:50 for 3 days at RT, and 
> secondary 1:200 for 2 days at RT.  Despite these long incubations I'm 
> having trouble with antibody penetration, and am starting to look into 
> permeabliziation methods.  I see several techniques in the literature: 
> enzyme digestion, adding Triton-X 100 and/or DMSO to the washing and 
> incubation buffers, and incubations in acetone or methanol after 
> fixation, but there's no explanation (that I can find) about how these 
> techniques work.  Does anyone have experience with these issues?  What 
> does DMSO or Triton-X or acetone "do" to the tissues?  What kinds of 
> concentrations have you found useful or not useful?  There is some 
> indication of a protective, very very thin "sheath" of tissue around 
> the actual neurons of the brain.  If this is the case, is enzymatic 
> digestion of the tissue the only option?  Pros or cons of the 
> different treatments?
>
> I know this is a long and detailed question.  Thanks so much for any 
> help you might provide.
>
> Mario Muscedere       
> Biology Department
> Boston University
> 5 Cummington Street
> Boston, MA, 02215 
> Office: 409 BRB
> Office #: 617-353-6977
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>


-- 
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff@umdnj.edu
**********************************************

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