Re: [Histonet] CD4/CD8 staining in murine lungs

From:Gayle Callis


Our lung speciality laboratory does not use peroxidase methods with lung, 
they get too much background even with endog peroxidase blocking, they 
prefer AP method instead and with Permanent Red from DAKO, superb, 
sensitive and great contrast with hematoxylin NBT/BCIP, as sensitive as 
DAB, but use nuclear fast red as the counterstain with this one.   TBS is 
the buffer with 0.05% Tween 20 for AP method.

We fill lung with OCT, cut 5 um frozens, fix with acetone/alcohol (I will 
be glad to provide more detail on this fixative,  but it is also already in 
Histonet archives many times)  The CD4 and CD8 clones come from BD 
Pharmingen, and we only use either goat or Donkey F(ab')2 frag of IgG anti 
Rat -biotin, adsorbed to mouse,with these antibodies to avoid cross 
reaction to fc receptors and between closely related species.  We also have 
superb results when both of these antibodies are already biotinylated. then 
come back with Strepavidin-HRP, no secondary and less time.

We also use a 10% goat or Donkey serum (depends on host of secondary used) 
with 2.5% mouse serum as the normal serum block 30 min,  and use this block 
as the diluent for the secondary antibody OR for a biotinylated Rat 
antiMouse CD4 or CD8, trust me, it works.

We prefer to do our own inhouse dilutions of Strepavidin-HRP or AP from 
Biosource, although Southern Biotechnology has excellent reagent, 0.5 - 0.6 
mg/ml diluted 1:500, for 20 min

Be sure you develop your DAB using microscope to control overdevelopment 
and do a dilution panel for both antibodies starting at 10 ug/ml.  Our CD4 
is at 1:500 or so, a 0.5mg/ml conc, and the CD8 is 1:200, always the 
weakest of the two.  Different chromogens wil have different conc of 
primary, so be sure to do the dilution panel.  Secondaries are generally 2 
- 5 ug/ml.

At 12:23 PM 8/24/2006, you wrote:
>    I am new to the world of Frozen section IHC and am trying to 
> standardize a protocol for cd8/cd4 staaing in murine lung tissues 
> embedded in OCT.  I have tried without success to stain these sections 
> with primary antibodies from biolegend, secondary antibody biotinylted 
> anti-rat IgG(H+L) from vector labs.  I was wondering if anybody has 
> recomendations on a protocol or better antibodies than the one I am 
> using.  Also, in developing my slides with DAB (biogenex), sometimes my 
> whole tissue sections turns brown within the matter of seconds.  I have 
> done through washes to remove any unbound antibody.  I don't know why or 
> where this is occuring.  Is it in my snap freezing, sectioning, or 
> staining?  Thanks in advance.
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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