RE: [Histonet] mouse spleen confocal

From:"Favara, Cynthia \(NIH/NIAID\) [E]"

Hopefully Gayle Callis will reply she is the guru, she has written
extensively and I am sure you can find information if you do a search of
the archives.


Cynthia Favara
903 South 4th Street
Hamilton, MT 59840

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-----Original Message-----
From: Scott Parker [] 
Sent: Tuesday, April 18, 2006 9:05 AM
To: histonet
Subject: [Histonet] mouse spleen confocal

Dear all,
Can anybody help me with some antibody staining I have been doing on
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
we see is vast amounts of auto-fluorescence making antibody detection
impossible. We really need to get this sorted because ultimatley we'd
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)
stored at -20.
When ready for use slides are warmed to room temp and fixed in -20 C
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
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
be used. Furthermore I have tried fixing in 3.2 paraformaldehyde with
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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