[Histonet] immunofluorescent antibody tissue controls

[Tony Henwood]

Becky,
>From our manual:

Document Procedure:		
Immunofluorescence  Frozen Section


Principle:	
Immunofluorescence is part of immunohistochemistry that combines the basic
principles of histochemistry with the high degree of molecular specificity
inherent in the antigen-antibody reaction.

In tissue there are essentially two forms of fluorescence, primary or
autofluorescence that can be seen in collagen fibres (blue green) or in
lipids (shades of yellow) and secondary fluorescence where fluorochrome dyes
applied and the created immuncomplex fluoresces when activated by UV light.
Localisation is revealed by labelling one of the components of the staining
reaction with a fluorochrome dye. Flourescence isothyocyanate (FITC) is the
commonly used dye for this purpose.

Purpose:	To demonstrate the presence of IgA, IgG, IgM, C3c, C1q and
Fibrinogen antigens in skin lesions and renal biopsies by employing
fluorescence technique instead of immunoperoxidase.
Controls: Not used with frozen specimens	
Reagents:	

1. Tris Buffer - as used in immunoperoxidase

2. Antibodies:
a. IgG-FITC			Dako  F0202
b. IgA-FITC			Dako  F0204
c. IgM-FITC			Dako  F0203
d. C3c-FITC			Dako  F0201
e. C1q-FITC			Dako  F0254
f. Fibrinogen-FITC		Dako  F0111

Dilute antibodies 1/10 in Antibody diluent

3. 1% Toluidine Blue

4. Permafluor mounting medium

Procedure:

1. Freeze tissues as rapidly as possible.

2. Trim and cut first section, stain with 1% Toluidine blue to assess the
presence of glomeruli

3. Cut 7 sections at 5 microns, fix 7th section in methanol for H&E staining


4. Air dry sections 15minutes.

5. Place sections in Tris buffer at 37oC for 7 minutes

6. Apply diluted antibodies to each section and incubate at room temperature
60min. Cover slides from light

7. Place slides in Tris buffer at 37oC for 7 minutes.

8. Apply drops of Permafluor & coverslip - dry for 20 mins.

Slides mounted with Permafluor are stable at RT -Long term storage at 4°C.

Reference:	

Henwood, Harmata & Scott, (1983)  "A Control for Routine Immunofluorescent
Histochemistry". Aust J Med Lab Sc 4:187-188.

Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html



-----Original Message-----
From: Garrison, Becky [mailto:becky.garrison <@t> jax.ufl.edu]
Sent: Friday, 6 February 2004 8:08 AM
To: 'LINDA MARGRAF'; Histonet <@t> lists.utsouthwestern.edu
Cc: MICHELLE MCNEESE
Subject: RE: [Histonet] immunofluorescent antibody tissue controls


Linda, 
Since we on an immunofluorescent thread...would any one
be willing to share their protocol for IF on kidney biopsies
(IgG, IgA, IgM, C3) including source/dilutions of antibodies?
Thanks.

Becky Garrison
Pathology
Shands Jacksonville
904-244-6237

-----Original Message-----
From: LINDA MARGRAF [mailto:LINDA.MARGRAF <@t> childrens.com]
Sent: Thursday, February 05, 2004 1:27 PM
To: Histonet <@t> lists.utsouthwestern.edu
Cc: MICHELLE MCNEESE
Subject: [Histonet] immunofluorescent antibody tissue controls


Dear Histonetters
We are continually running low on control tissue for our
immunofluorescent (IF) stains for IgG, IgA, IgM, and C3 that we run on
kidney biopsies.  Our pediatric population rarely has a kidney removed
for diseases with antibody deposits and none of our neighboring
hospitals has any control tissue to spare. DOes anyone know of a good
commercial source for positive IF control tissue for Igs and C3?
Thanks
Linda M
Histonet administrator
Director of Anatomic Pathology
Children's Medical Center of Dallas


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