RE: [Histonet] autofluorescence?

From:"Charles Scouten"

Since red blood cells are notorious for autofluroscence, I would guess
they are indeed blood vessels, although I have not worked with that
material.  Is there any way you can sacrifice perfuse the tissue before
sectioning?  Can you get a needle into a beating heart at that stage?
Wash out the red blood cells, and the problem should go away.

Charles W.  Scouten, Ph.D. 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300 x 342
FAX  314 522 0377 

-----Original Message-----
[] On Behalf Of yasushi
Sent: Thursday, May 11, 2006 11:13 AM
Subject: [Histonet] autofluorescence?


We are doing immunohistochemistry using Cy2, Cy3 and Cy5-conjugated
secondary antibodies on 20um-thick cryosections of embryonic mouse
brains. Especially with Cy2- filter setting, we sometimes see a number
of auto-fluorescence-like signals within the brain sections.  
They are thin and long, kind of looking like blood vessels. They show up
even when we do not use Cy2-conjugated secondaries. The same problem has
happened to Cy3-filter setting, too, although less frequently. I wonder
if anyone has a idea about what they are and how to prevent them from
showing up.

We fix brains in 4%PFA/PB overnight, wash and cryoprotect in sucrose/
PB, and freeze in OCT in plastic molds on petri dish on liquid nitrogen.
We cut 20um cryosections, dry them for an hour or so, and do regular
antibody staining, do DAPI staining, dehydrate, and mount in DPX.

Thank you for your help.

Yasushi Nakagawa

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