[Histonet] DIF tissue in GLUT

[James L Burchette]

Dear Anne,

I assume the glutaraldehyde tissue has been processed and embedded in 
paraffin. Following removal of paraffin and any pretreatment to unmask the 
antigen, you can block autofluorescence or change the color by treating 
the sections with 0.05% sodium borohydride prepared in PBS for 30 minutes. 
After the borohydride treatment, carefully wash the slides with multiple 
changes of PBS for thirty minutes. I would also use a good quality silane 
treated slide for maximum tissue adhesion.
A comment of caution: sodium borohydride is poisonous. Be extremely 
careful with the powder.

Reference for NaBH4:
Embedment in Glycol Methacrylate at Low Temperature Allows 
Immunofluorescent Localization of a Labile Tissue Protein. JA Carnegie, ME 
McCully and HA Robertson, Journal of Histochemistry and Cytochemistry, 
Vol. 28, No. 4, pp 308-310, 1980

Methods in Cell Biology, by Leslie Wilson, page 117, section VI, Use of 
Glutaraldehyde in Immunofluorescence Microscopy. Academic Press 1982

I used this method in a double label fluorescence project a few years ago 
with excellent success.

23.     Endothelial Metaplasia in the Iridocorneal Endothelial (ICE) 
Syndrome. DN Howell, T Damms, JL Burchette, JR Ross, WR Green. 
Investigative Ophthalmology and Visual Science, Vol. 38, No. 9, pages 
1896-1901, August 1997.

Best Regards,
Jim

Jim Burchette, HT(ASCP) QIHC
"A simple histotech from a little country hospital in North Carolina"



Anne van Binsbergen <annigyg <@t> gmail.com> 
Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
08/27/2009 06:27 AM

To
John Kiernan <jkiernan <@t> uwo.ca>, "histonet <@t> lists.utsouthwestern.edu" 
<Histonet <@t> lists.utsouthwestern.edu>
cc

Subject
[Histonet] DIF tissue in GLUT






Dear John and other knowledgeable Histonetters and Gurus of the trade....

Renal bx tissue core has been placed in Gluteraldehyde/Trumps EM
fixative by mistake (we all make mistakes) - we now need to do DIF
Does anyone out there have a method for this?

-- 
Anne van Binsbergen (Hope)
Abu Dhabi
UAE
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