[Histonet] mouse spleen confocal

From:"Scott Parker"

Dear all,
Can anybody help me with some antibody staining I have been doing on mouse
spleen? We are trying to detect CD8 in 5 um sections using CD8b.2 (ly-3.2) (
53-5.8) with secondary Alexa Fluor 633 from mol. probes. Unfortunately all
we see is vast amounts of auto-fluorescence making antibody detection
impossible. We really need to get this sorted because ultimatley we'd like
to have GFP and two different secondaries (labelling 3 things in total).
Our protocol is basically:

Spleens are removed to OCT and frozen in isopentane, sectioned (5 um) and
stored at -20.
When ready for use slides are warmed to room temp and fixed in -20 C acetone
for 3 mins then re-hydrated in PBS for 5 mins.
Sections are allowed to dry and blocked for 30 min in 10% normal mouse serum
in PBS (blocking buffer).
Sections are stained in blocking buffer, washed and then stained with
secondary (also in blocking buffer). We wash in PBS with 0.1% tween.
Having done some searches on histonet I see that tween should probably not
be used. Furthermore I have tried fixing in 3.2 paraformaldehyde with the
same result.
Sections that are treated as above without antibody look exactly the same as
sections treated with antibody when viewed under the confocal. Basically
lots of auto-fluorescence nicely labelling all the cells.
Any help or protocols would be glady appreciated.

Scott Parker, Ph.D.
School of Medicine
St. Louis University
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