[Histonet] RE:BrdU and GFP staining

[Johnson, Teri]

Steve wrote: >>We have several FFPE blocks from 2003 that express GFP.
Our lead scientist would like to perform Brdu IHC in the same slides
hoping to express both.  As expected the AR removes the GFP green label.
Has anyone any experience expressing Brdu and GFP on the same slide.

First question I have is - does the fluorescence of the GFP in your
tissues survive the paraffin processing? Some labs can demonstrate it,
but most can't. I think the secret to success is using a processing
temperature that stays at 58 degrees C or below. Low melt point
paraffins (of which we have none) are supposed to be superior for
post-paraffin processing/embedding direct visualization of GFP
fluorescence.

In her response, Sarah gave great advice and information, especially
regarding the limitation if you expect co-localization.

Since the BrdU antibody requires denaturing of some sort, multiple
immunostaining techniques (especially fluorescence) can be very tricky.
We use the BrdU antibody from Amersham, and the working solution
contains DNAse enzyme which may or may not work in a cocktail with
another antibody.

Our GFP does use HIER (antigen retrieval in citrate buffer) for
pretreatment, as does our BrdU. If I were to try this, this would be my
ideal approach on mouse tissues:

Do single staining first on the tissues using each antibody separately
to make sure you get immunostaining using this detection scheme. In
parallel, run slides with tissues known to be negative for GFP and BrdU
as specificity controls. If all looks good, proceed with double stain
protocol.

Here's what would probably work in our lab:

HIER as usual, for us 10' @95 degreesC in a microwave
Block as usual with casein-type blocking reagent, 10'
Apply cocktail GFP and BrdU antibodies: rabbit anti-GFP (Novus
Biologicals, NB 600-303) at 1:500 + mouse anti-BrdU (Amersham, prepare
stock as recommended, but dilute out to 1:50 for working concentration)
- incubate 1 hour at room temperature
Secondary antibody cocktail - Alexa fluor 488 donkey anti-rabbit 1:300
(GFP label) + Alexa fluor 568 anti-mouse IgG2a 1:300 (BrdU label) - 30
minutes - 1 hour at room temperature
DAPI counterstain

Run one negative using non-immune serum cocktails for the primary
antibody cocktails, everything else the same.
Run one slide using above protocol.

Verify signal by comparing to your single IHC techniques, and
troubleshoot if necessary.

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110