murine CD4/8, reply

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From:Gayle Callis <> (by way of histonet)
To:histonet <>
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I agree with Jamie Erickson.  We use PharminGen antibodies with same
concentrations, frozens (our fixation is a bit longer with acetone)
but results are excellent.  We prefer F(ab)'2 secondaries, mouse and
human absrbed as secondary, but a new twist to using a secondary,
and it does work! is to use a mouse antirat-IgG (H+L) F(ab)'2 biotinlyated
secondary, 1:250 diluted in PBS with 0.1% BSA (TPBS also
works) and original article has 0.05% Tween 20 added to buffer, we
took it out.  Jackson ImmunoResearch was source of latter secondary, and we
had some of the cleanest staining ever on a rat antimouse inhouse antibody.

The primary is diluted in buffer, but 25ug/ml Rat IgG is added to
this diluent.  The rationale is there will be no cross reaction
with mouse B cell immunoglobulins, and it works, normal serum blocking
is not necessary.  Dako peroxidase block is recommended by PharminGen,
avoid methanol.   When we use a goat antirat secondary, it is diluted
in the normal blocking serum (2.5% mouse/5 - 10% goat serum)

For paraffin sections and NO ANTIGEN RETRIEVAL,  you can try a
nonformalin fixative H Nitta et al, Cell Vision, vol 1, no 4, 1997.
it is zinc chloride, zinc acetate, calcium acetate in TRIS-HCl buffer,
and gives excellent staining with dilutions comparable to frozen
section/acetone fixtion.  Is a wonderful way of not struggling with
NBF/fixed mouse tissues.  Will be glad to email you the recipe.

Gayle Callis

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