Re: accepted fixation protocols

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From:Tim Morken <>
Date:Tue, 16 Mar 1999 09:17:14 -0500 (EST)


The use of fluorescent tags predates the use of the enzyme-chromogen 
systems we use today in paraffin. Using a fluorescent-tagged primary 
antibody was the most efficient and easiest method at the time (and 
still is in many cases). Antibody avidity and sensitivity is better on 
non-formalin-fixed tissue and the fluorescent tag is quite sensitive. 
Some antigens are only accessable in frozen tissue.

Fixation of tissue with formalin will cause some problems with 
autofluorescence so fluorescent tags are usually used on frozen 
sections. You can use an unstained section as a control for 
autofluorescence. Yo may also be able to get a narrow-band filter that 
filters out most of the autofluorescence. Some people have used powderd 
milk as an additive to prevent autofluorescence.

Since your tissues are already fixed, however, you may as well go to a 
multi-step system, which will give you more sensitivity and a more 
options for detection. 

Something you can try is to use a system in which you use a secondary 
anti-FITC (or whatever fluorecein you are using) and then a third step 
of either a biotinylated antibody or PAP to get an amplified link (you 
may be able to use a biotinylated link on the primary but sometimes the 
fluorecein label prevents this method). This would eliminate the need 
for fluorescence and get you into a HRP or alk phos system and chomogen. 
In those cases autofluorescence would cease to be a problem.

Tim Morken, B.A., EMT(MSA), HTL(ASCP) 
Infectious Disease Pathology
Centers for Disease Control
1600 Clifton Rd.
Atlanta, GA 30333


FAX:  (404)639-3043

----Original Message Follows----
From: Judy Trogadis <>
To: (histonet)
Subject: accepted fixation protocols
Date: Mon, 15 Mar 1999 12:10:32 -0500 (EST)

Dear Histonetters:

I am trying to stain human lens sections, first with fluorescent markers
for identifying fiber cells and nuclei, then with other antibodies to
study cataract formation.

The tissue is human archival eyes, formalin fixed and paraffin embedded. 
So far, the results include mostly autofluorescence. My question is the 
following: Is it the accepted convention that paraffin sections are used
for immunohistochemistry and cryosections for immunofluorescence? That 
my impression from doing a literature search. The other explanation may
be that routine clinical labs use paraffin embedding and 
because it is more common (?faster, ?easier) and immunofluorescence is
more of a research tool, although both disciplines use cryosections.
Any opinions?

Unfortunately, the most critical antibody I want to use ultimately so 
has only worked with immunofluorescence so I will have to try quenching
the autofluorescence. How realistic is that with lens, especially 
cataractous lens?

If anyone knows tha answers to these questions, it is this group, so
thank you in advance.


Judy Trogadis
Eye Research Institute and
University of Toronto
Toronto Hospital, Western Div.
399 Bathurst  St.
Toronto, Canada M5T 2S8

phone: 416-603-5088
Fax:   416-603-5126

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