[Histonet] DIF procedures

[Katri Tuomala]

Ron,
we have used a simple direct IF method for years to do IgA, IgG, IgM, 
complement3c, fibrinogen, albumin, kappa and lambda on fresh frozen human 
renal biopsies.
We collect the needle biopsy at the ultrasound department, when the biopsy 
is being taken. One core is put in 10% NBF, second one snap frozen and a 
small piece is fixed in 2% glutaraldehyde for EM.

When IF is requested by the pathologist, we cut 5mu sections from the frozen 
piece and let slides ( use poly-l-lysine or + slides) dry at room 
temperature for minimum 30 minutes. H&E gets done on one slide to make sure 
there are glomeruli in the biopsy. We then do the direct immunofluorescence 
on unfixed sections:
1. Wash in PBS for 15 minutes
2. Apply fluorescein conjugated antibody on the sections for 30 minutes:
Dako FITC/IgA at 1:20 in PBS
Dako FITC/IgG at 1:30
Dako FITC/IgM at 1:20
Dako FITC/C3c at 1:50
Dako FITC/fibrinogen at 1:10
Dako FITC/kappa at 1:20
Dako FITC/lambda at 1:20
Dako FITC/albumin at 1:30
(keep slides away from light, cover with a towel)
3. Wash well in PBS x2 for 15 minutes each.
4. Coverslip with aqueous non-fluorescent mounting medium, store in fridge 
until ready to read. Photographic record is made of results, since these 
preparations are not considered permanent.

Hope this helps,
Katri

Katri Tuomala
Hamilton, Ontario, Canada

----- Original Message ----- 
From: <pathrm35 <@t> adelphia.net>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Tuesday, July 26, 2005 10:55 AM
Subject: [Histonet] DIF procedures


> Fellow Techs,
> Does anyone have Direct Immunofluorescence procedures they are willing to 
> share? We are interested in CD3, fibrinogen, IgA, IgM and IgG.
> Thanks in advance.
> Ron Martin, BS HT (ASCP) HTL
> fax 561-721-1249
>
>
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