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

From:"Johnson, Teri"


Quite the dilemma!  Thanks for your detailed information on what you're
seeing and what you've tried.  I find this question interesting because
at the moment I am sifting through a similar dilemma with regards to
immunofluorescence and chromogenic immunostaining on mouse intestine.

We have done immunostaining using an anti-BrdU mouse monoclonal antibody
in mouse tissues. Routine technique includes 3% H2O2 for 10 minutes to
block endogenous peroxidase, PowerBlock (biogenex) as a blocking reagent
prior to primary antibody incubation, and avidin/biotin block (Vector)
when using any detection involving biotin. 

Using an anti-mouse secondary antibody gives us strong staining with
some intensely stained cells in the lamina propria, even in the negative
sample (non-immune serum).  We see this on both chromogenic (DAB) and
fluorescent immuno procedures.  Thinking it was a reaction with the
anti-mouse secondary antibody (plasma cells or mast cells), we plugged
this BrdU antibody in to the Dako ARK kit (biotinylates the primary +
adds blockers and uses streptavidin/HRP) and also the Innogenex mouse on
mouse kit.  Believe it or not, we still got strong staining of these
same cells on the negative control and also on the BrdU antibody sample.
At the same time, we also tried using an anti-IgG2a anti-mouse/HRP
labeled secondary antibody, and in the negative control there was no
staining, while in the BrdU antibody sample there was a notable
reduction in the number of positive cells stained in the lamina propria.
Since we're using DAB chromogen and not fluorescence for this particular
study, I don't think the SIF cell explanation fits here. This phenomena
is not completely due to anti-mouse secondaries because we still see the
reaction with the biotin/streptavidin detection.  It isn't endogenous
peroxidase because it is eradicated using an isotype-specific secondary
on negative control, however we do see reactivity when the BrdU antibody
is used.

Consequently we've adopted using the anti-IgG2a secondary antibody as a
"cure" to this problem, but I'm still curious about what's really going
on!  Perhaps it's a sensitivity issue with regards to the detection
scheme (negative controls), but the isotype specific secondary antibody
gives us staining in the BrdU antibody samples (specific staining?)  I
am in search of answers to this dilemma, so if anybody has any
suggestions, I'm open to hearing them.  

I suspect you're seeing the same cells staining that we are, but
additionally you have some background issues with connective tissue and
muscle.  Aldehyde-fixed tissue tends to give high auto-fluorescence,
especially in muscle and red blood cells.  You might try using the
combination of acetone/alcohol (3 parts/1part) as a fixative for your
cryosectioned tissues to improve morphology and take the aldehyde
fixation out of the picture.  Here is the technique from Gayle Callis: 
An acetone/alcohol fixation (AA)  Air dry frozen sections overnight at
RT, they MUST 
BE VERY DRY.  Fix next day (of staining) in 75% acetone/25% absolute 
ethanol or 75 ml acetone mixed with 25 ml 100% ethanol (do NOT use
or any other alcohol) .  Fix for 5 minutes, then go directly to buffer 
rinse.  DO NOT air dry sections again, and proceed to staining.

Otherwise you might try any of the published techniques for abolishing
auto-fluorescence.  Many anecdotal reviews are mixed on how effective
these really are.

And, for the record, immunostaining with primary antibodies made in goat
is one of my LEAST favorite things to do. I would recommend
concentrating on using the rabbit polyclonal antibody for future

Would a silver reaction (special stain) work for your purposes to
identify these cells instead?

Best wishes,

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133

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