[Histonet] Immunofluorescence

Gayle Callis gayle.callis <@t> bresnan.net
Sat Dec 15 16:33:57 CST 2007


You didn't say how thick the sections will be?

Go to www.IHCworld.org  and click on the fluorescence area, then 
autofluorescence and how tissues sections can be treated to reduce this 

 One thing that can be done is USE the autofluorescence as a 
counterfluorescence i.e. like a counterstain.  If the tissue is prefixed 
with NBF or paraformaldehyde, then aldehyde induced autofluorescence 
results.  If she uses a fluorophore that is the opposite color of the 
autofluorescence, say that is greenish yellow, then a fluorophore that is 
red works much better.  Also a fluorophore that is near infrared region 
helps too, although one cannot visualize this with the human eye.

Be sure the fluorophore is the brightest possible, and resistant to 
photobleaching.  Recommended is Molecular Probes/Invitrogen Alexa 594 (an 
equivalent to Texas Red).  One can conjugate your own antibody or buy 
secondaries conjugated to this flourophore.

There are chemical treatments to reduce autofluorescence on aldehyde fixed 
tissues, even after fixation and before sectioning or on the section itself. 
One method uses 100 mM glycine treatment and this has been discussed on 
Histonet, check out the archives.  If the tissue is vibratomed in fresh 
state, then using a red fluorophore conjugated antibody can give some 
spectacular results.  One lab here vibratomes fresh lung tissue (approx 100 
um thick sections) containing cells labeled with Texas Red antibody.  The 
fresh tissue autofluoresced green, cells red, and they did CLSM z stacks 
with great success using an inverted microcope with a Zeiss CLSM.

If one can avoid aldehyde fixation, that helps but that is not always the 
case.  There is also a confocal listserver out of Buffalo, similar to 
Histonet that can be invaluable for more specific information when using the 
confocal for z stack imaging.  Have her subscribe to that and also get into 
their archives.  An excellent listserver.

Good luck

Gayle Callis

----- Original Message ----- 
From: "Cheryl Crowder" <ccrowder <@t> vetmed.lsu.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Saturday, December 15, 2007 8:04 AM
Subject: [Histonet] Immunofluorescence

> Hello - I have a researcher who has done IHC on her tissues with good
> success.  Now she is trying to do fluorescent antibodies on thick sections
> to be able to do 3D imaging.  I have no experience with this technique. 
> Her
> problem, tremendous background staining.   Can anyone give us any tips for
> reducing this staining?   Thanking you in advance,
> Cheryl
> Cheryl Crowder, BA, HTL(ASCP)
> Chief Technologist
> Anatomic Pathology
> Department of Pathobiological Sciences
> School of Veterinary Medicine
> Louisiana State University
> Skip Bertman Drive
> Baton Rouge, LA  70803
> 225-578-9734
> FAX:  225-578-9720
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet 

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