[Histonet] Immunofluorescence problem-Non-specific binding appears incontrol slide

[John Kiernan]

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.
______________________________

"Stylopoulos, Nicholas" wrote:
> 
> Hello everybody and thank you for taking the time to read this message. I have
> been struggling with immunofluorescence and I have the following problem: My
> control slide (secondary antibody only) appears to have specific staining. I
> have repeated the assay many times (Also, I showed my control slide to many
> people and they asked me whether I am sure that I have not added my primary
> antibody as well!). My primary antibody is goat anti-rat (against chromogranin
> A). I have also tried rabbit anti-rat. My secondary is donkey anti-goat FITC
> conjugated (I have tried donkey anti-rabbit and goat anti-rabbit).  Basically, I
> am trying to visualize the endocrine cells in the gastrointestinal tract. This
> is my protocol:
> 
> Tissue: rat ileum-snap frozen in isopentane
> 
> Sections: Frozen sections 3-5?m
> 
> Fixation: 4% paraformaldehyde (I tried also acetone -20C from 30secs to 20min
> and it seems that acetone destroys the histology of my sections)
> 
> Washx3 with filtered PBS 1X
> 
> Triton x-100 0.05% for 5min
> 
> Wash x1 with filtered PBS 1X
> 
> 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 (from Innovex too) or sodium azide 0.1%)
> 
> Primary Antibody in PBS for 1hr
> 
> Washx3 with filtered PBS 1X
> 
> Secondary antibody in PBS for 1 hr
> 
> Washx6 with filtered PBS 1X
> 
> Mount with DAPI
> 
> 
> 
> Any suggestions will be extremely appreciated....Thanks again!
> 
> 
> 
> Nick
> 
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