[Histonet] Immunofluorescence problem-Non-specific binding appears in control slide

[Stylopoulos, Nicholas]

Thank you so much for all your suggestions! 

I found the papers on Falck-Hillarp reaction that Al [Floyd] suggested. It
sounds that it may be possible to be the cause of the specific-looking
autofluorescence, because my specific binding occurs (mostly) in the lamina
propria of almost each single villus and the outer layers of my specimen (in the
connective tissue and the muscular layer). This is very similar to what John
[Kiernan] suggested in his response. This "specific binding" is not related to
the nature of the secondary antibody (FITC, TRITC, anti-goat or anti-rabbit) and
the concentration (I tried dilutions up to 1:5000). I can even see very nice
"rings" around the nuclei (like something is stained in the ER-Golgi). Also it
is very similar to the picture I get when I add my primary (which is
anti-chromogranin A). It looks like almost the same cells are stained! As a
matter of fact, I thought that I had cross-contamination, so I started to use
filtered tip pipettes and prepare new reagents every time I would do the assay.
The only two arguments against the Falck-Hillarp reaction are that 1) the
fluorescence is prominent only after I add my secondary antibody and 2) I tried
fixing the tissues with Acetone and I got a slightly better picture but not
extremely different. However, I did not try acetone many times because it would
destroy the histology and additionally the DAPI staining was kind of diffused
and not very helpful (which I assume is expected when acetone is used as a
fixative). Finally, to answer Julia's [Edgar] question, I did try an anti-rabbit
secondary which did not give me a different picture. 

In the next few days,  I would like to rule out the Falck-Hillarp issue and it
would be amazing if you could give me some info on the following two questions:

1)      I snap-freeze the tissues in isopentane. Is it possible that isopentane
might create the problem? The reason I am asking is that -from what I read- in
the initial description of Falck-Hillarp method, the tissues were snap-frozen in
propane.

2)      What fixation method would you suggest instead of paraformaldehyde? Is
is possible to use Acetone but improve the histologic picture? Are there any
other methods that you would suggest? I have already harvested the tissues after
I snap-frozen them in isopentane (and some in liquid nitrogen).

 

Thank you again for your help!!! 

 

Nick

 

 

 

 

Nicholas
Do you get the same problem with the anti-rabbit antibodies? Anit- goat can
be very 'dirty' I find.
Julia
Julia Edgar BSc (Hons), PhD
Applied Neurobiology Group
University of Glasgow Veterinary School
Bearsden Road
Glasgow
G61 1QH
Tel: 0141 330 5818
e-mail: je22r <@t> udcf.gla.ac.uk


Al Floyd's astute suggestion of a Falck-Hillarp
reaction is certainly a possible cause of 
"specific-looking" autofluorescence. In rat intestine
and its associated connective tissue you can expect
a F-H reaction in mast cells and classical argentaffin
cells, from their high content of serotonin. (The
noradrenaline in sympathetic axons doesn't give a
localized fluorescent product with ordinary
formaldehyde solutions.)

If your unwanted fluorescence is definitely due to
binding of the FITC-labelled secondary antiserum, a
possible cause may be binding of the FITC-secondary
to something in your "blocking" protein:

You wrote:
> ... Block with 10% donkey serum (I tried adding 10% 
> rat serum or 3% BSA) for 2hrs (I tried also blocking
> overnight... or I added Background buster (from
> Inovvex) and Fc receptor blocker ...

Not all your  "blocking" substances are identified,
but they are all intended to bind to the tissue.
Rat serum may not be good stuff to put on a rat
tissue if the secondary antiserum is an
anti-(rat immunoglobulin).

For competitive blocking of low affinity binding of
primary and labelled antibodies it's probably best
to go with either a "generic protein" such as bovine
albumin or a non-immune serum from the host species
of the secondary. Before using any trade-name product,
find out what it is and how it may work. Follow up
aome of the references in the small print at the
bottom of the brochure.

Clear thinking (what sticks to what, and why?) is
the paramount principle in immunohistochemistry.

John Kiernan
Anatomy, UWO
London, Canada.


Hello Nick,

You may be running into the problem of Falck-Hillarp reactive cells, more
commonly referred to as "SIF" cells.  SIF stands for Small Intensely
Fluorescent cells.  This technique was quite popular back in the 1960's.
In brief, any cell that contains epinephrine, nor-epi, or Dopamine will
become very highly fluorescent after exposure to formalin.  In the actual
Falck-Hillarp technique, the exposure is to gaseous formaldehyde vapor,
but you may be getting some of the same reaction with your procedure.
Generally, Epi and Nor-epi fluoresce with a color that is very similar to
flourescein, while Dopamine has a more orange color. 

There is actually a monograph available titled SIF cells.  I forget the
exact date, but think it is early '70's.  I have a copy on my bookshelf,
but at the moment am a long way from the bookshelf!

Try a Google, Google Scholar or PubMed search for SIF cells - you should
find quite a few publications.  It was a very popular technique before
the days of immunostains.

Al Floyd
Alton D. Floyd, Ph.D.
23126 South Shore Drive
Edwardsburg, MI 49112
Cell: 574 215-0703