Re: accepted fixation protocols

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From:tylee <>
To:Tim Morken <>,
Date:Tue, 16 Mar 1999 18:38:57 -0600
Content-Type:text/plain; charset="iso-8859-1"


I'm not an expert on fluorescence; but, can't you avoid autofluorescence by
using "redder" (i.e longer wavelength) fluorescent reporters (rhodamine or
others) to avoid those "greenish" wavelengths where inherent
autofluorescence occurs?

I think remembr reading in the Molecular Probes handbook about this effect
some time ago.

Ty Lee
-----Original Message-----
From: Tim Morken <>
To: <>
Date: Tuesday, March 16, 1999 10:54 AM
Subject: Re: accepted fixation protocols

>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
>Get Your Private, Free Email at

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