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

From:Gayle Callis

Teri and Nick,

Just curious about which block you use with Strepavidin-Biotin methods?  We 
use the Strepavidin/biotin block from Vector instead of their avidin/biotin 
blocker when working with Strepavidin-Alexa fluorophores and 
Strepavidin/biotin for IHC.

I asked the Vector technical service contact and this was his reply (some 
time ago)

         Thanks for the email. Streptavidin does have some subtle binding 
         than avidin. Streptavidin has been shown to bind to fibronectin 
         that are expressed on cell types. These integrins (extracellular 
         receptors) mediate cell adhesion, migration etc and streptavidin 
binds to
         these too. So the bottomline this that streptavidin based 
detection systems
         may give background/non-specific staining due to this inetgrin 
binding that
         is not blocked by a standard avidin/biotin blocking step. Hence 
the idea
         behind the streptavidin/biotin kit. See the following reference:
         Alon, R. et al (1991) Cell-adhesive properties of streptavidin are 
         by the exposure of an RGD-like RYD site. Eur. J. Cell Biol. 

         The nomenclature in this article is dated. The GpIIbIIIa is now 
known as the
         alpha 5 beta 1 dimer that specifically binds fibronectin via the 
RGD amino
         acid sequence. There are antibodies available against the alpha 5 
         itself. Migratory cells, such as embryonic cells, some tumor cells 
etc would
         be high expressors of this integrin. Obviously you could either 
block with
the streptavidin kit or use an avidin based detection system.

*** We have found the Strepavidin-biotin blocking kit works well, things 
remain clean with both IFA and IHC.

Teri made a good suggestion to use a secondary against the specific isotype.

With IHC, have you thought of using the glucose oxidase endogenous 
peroxidase blocking method, very thorough and kills both peroxidases and 
pseudoperoxidases, works with frozen sections and with paraffin 
sections.  I will be happy to send this to you, we found it took out a 
cells staining nonspecfically positive in our  negative control (not good!) 
and something you don't want when the target cell population is sparse to 
very fee then you want NO spurious nonspecific staining of any 
cells.  GLUOX as we called it cleaned up a multitude of staining sins.

There will always be some autofluorescence in the tissue, even after 
acetone alcohol or acetone fixation of frozen sections.  One thing to 
consider is using a fluorophore that contrasts the autofluorescence, in red 
rather than FITC colors - we use Alexa 555 to do this, then 
autofluorescence becomes a counterfluorescence - making interesting 
photos!!!   The strength of the signal of your fluorophore will help, 
something that is extremely bright (Alexa 488 versus FITC)  and overcomes 
some of the autofluorence also helps.  We also are picky where we purchase 
our fluorophore labelled antibodies, by F(ab')2 frag of IgG to avoid fc 
receptor binding in tissue, and careful adsorption to the mouse or species 
being stained.

We have never tried methods to abolish autofluroescence, in general, we 
live with it or deal with it in a different way.

At 10:28 AM 8/16/2005, you wrote:
>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
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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