[Histonet] question about immunofluorescence staining in
gayle.callis <@t> bresnan.net
Tue Mar 18 19:38:32 CDT 2008
----- Original Message -----
From: "Sharon" <scoop <@t> mail.nih.gov>
To: <Histonet <@t> lists.utsouthwestern.edu>
Sent: Tuesday, March 18, 2008 5:35 PM
Subject: [Histonet] question about immunofluorescence staining in
> Dear experts,
> My boss has asked me about immunofluorescence staining in mouse spleen:
> How do you get rid of fluorescent background in mouse spleen?
Use fresh snap frozen tissues (not cryostat freezing, but true snap
freezing) and cut at -17C on a cryostat, pick up on Plus charge, air dry,
fix with cold acetone or if doing CD markers, 25% absolute ethanol/75%
acetone at RT for 5 minutes but go directly to buffer after this fixative.
Then careful normal serum blocking and also clever use of primary
antibodies, and secondaries that are F(ab')2 frag of IgG adsorbed to mouse
tissue. Jackson has excellent secondaries. We also use biotinylated
primary antibodies and come back with Molecular Probes Streapavidin Alexa
conjugates, 488 or 594. Have to do avidin/biotin blocking though.
No autofluorescence other than tissue components that do autofluoresce.
I suggested ammonia ethanol quenching which I know works in bone marrow.
Is there a better method?
*A better method for FFPE tissues? There are ways, but not always
Also, my boss claims that the macrophages have autofluorescence (formalin
fixed paraffin embedded tissue).
*And a lot of other tissue components too. Aldehydes induce
autofluorescence, and it is hard to remove. Go to the IHCWorld website and
read up on how autofluorescence can be removed and also a wonderful tutorial
on fluorescent staining (link to Wright State?). We do not do murine CD
immunosfluorescent staining on formalin fixed tissues. There are some ways
to get around this too if CD work is not needed. Formalin fixation, no more
than 8 hours, paraffin sections, treat sections with 100 mM glycine in PBS
or TBS pH 7.4, 20 minutes, then use polymer kit with alk phos, and DAKO's
permanent red, underdeveloped a very light for fluorescence work. Also,
there is a lot of information in Histonet archives - have fun searching.
I always thought the rbcs would be the autofluorescent cells. Do the
macrophages have autofluorescence?
Finally, is it reasonable to do frozen sections of mouse spleen? I
thought frozen sections fixed in ice cold acetone or acetone-methanol
might solve the problem, but I was told that frozen sections would be much
to thick to get any good immunofluorescent pictures on.
We have no problems cutting 4 um frozen sections (5 um is preferred) of
mouse spleen from fresh snap frozen tissues as long as the tissue is
equilibrated to a cryostat cutting temperature of -17C. Do not come from a
cold bar to immediate sectioning, or the tissue will be crunchy. Thymus
can be cut at 3 um, and if careful, spleen could be too.
We do double IFA staining on 5 um FS of mouse spleen, can do triple,
without any problems. There are autofluroescent tissue components but the
fluorophores are so bright, the other dimmer components are not a factor.
What you were told was probably because they lacked the technics to do this
properly and without success. So go for it!
> Thanks for any info and suggestions.
> Unknowledgeably yours,
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