Re: [Histonet] Gr-1 staining in mouse spleen

From:Gayle Callis

GR-1 i.e. Ly-G6 Clone RB6-8CS should work very well with frozen sections, 
and it should stain in 30 minutes incubation time.  In fact, the CD4 and 
CD8 should stain in the same way with the same timing as suggested below.

However, endogenous peroxidase blocking using methanol is known to be very 
bad for lymphocyte CD type markers, I would use DAKO's peroxidase block in 
a buffer for frozen sections.

You need to do a dilution panel for the primary antibody, starting with a 
target concentration of 10 ug/ml and dilute out.  Your antibody is far to 
concentrated is you are using a direct application of 0.5mg/ml!!!   The 
recommended concentration for this antibody (found in specification sheet 
from BD)  is 1 to 5 ug/ml, which is a 1:500 dilution of the 
0.5mg/ml!!!!   So please note the comments in your protocol below.

At 11:54 AM 6/28/2006, you wrote:
>I have gotten wonderfully accurate information from histonet in the 
>past!  Now, I have been trying for Gr-1 staining of mouse spleen for 
>weeks.  The best result have come only by direct application of the BD 
>antibody at 0.5mg/mL and the results look like all of the white pulp cells 
>are positive.  Naive mice and tumor bearing mice look identical.  There is 
>nothing on my "no primary" controls.  However, I doubt this staining is 
>specific.  My protocol is as follows:
>*       Thaw slide mounted frozen sections (in foil pack), air dry, and 
>fix in acetone 10 minutes at RT.

Frozen sections should be totally dry, preferrably overnight, acetone 
fixation is fine 10 min at 4C, then air dry, go to buffer rinses.
Try 75% acetone/25% absolute ethanol for 5 min at RT, but go directly to 
buffer for 3 changes - you may find the morphology is better and you have 
excellent staining of the granulocytes.

>*       Air dry and apply PAP pen
>*       Hydrate in PBS (and removes embedding medium) the rest of the 
>protocol at RT
>*       Block endogenous peroxidase with 1:1:8 methanol:30% H2O2:PBS for 
>15 min.

Don't use methanol peroxidase blocking for murine CD markers,  use the DAKO 
peroxidase block for FS, 0.03% hydrogen peroxide or bring down the 
concentration of your hydrogen peroxide to 0.1% or even less diluted in 
buffer for 10 min

>*       Wash 3x PBS 5 min
>*       Permeabilize 30 min with 1.8% lysine. 0.2% triton X-100, 4% serum, 
>in PBS

You do not need to permeabilize, these are cell surface markers, and simply 
do a normal serum block of 10% serum matched to the host of the secondary 
antibody (which should be adsorbed to mouse).  Triton X-100 can remain but 
it should be used in all buffers, diluents, and you may want to reduce the 
concentration with a minimally fixed frozen section.  We add 2.5% mouse 
serum to our NSB when doing most of the rat antimouse CD markers.  That 
concentration can vary from 1 to 5% and also use this NSB to dilute your 
secondary antibody.

>*       Quick rinse in PBS

>*       Direct application of antibody BD# 553123 incubate overnight.

NO, this antibody needs to optimized with that dilution panel, and read the 
BD data sheet for recommended concentration, dilute the antibody in 5% 
normal serum matched to host of secondary antibody, you can use less for 30 

>*       Wash 3x PBS 5 min
>*       Vector biotinylated secondary #BA4001 rabbit anti-rat, 1:1000 
>dilution, 2 hours

Secondary antibody should have a concentration of 2 - 5 ug/ml, dilute in 
the normal serum block and incubate for 30 min at RT
We prefer to use goat or donkey antiRat (F(Ab')2 frag of IgG (0.5mg/ml) 
diluted 1:250 for 30 min at RT.

>*       Wash 3x PBS 5 min
>*       Vectastain ABC kit PK-4000 one hour

I presume this is the Elite kit, and you follow kit directions - 30 min is 
the recommended time??  at RT.

>*       Wash 2x PBS, 1x TBS,  5 min

Instead of having to change buffers use TBS instead of PBS throughout, much 

>*       DAB 0.05% + nickelous ammonium sulfate 0.5% in TBS 5 min
>*       Add 0.1% of 30% H2O2 for 5 min to develop black color

Watch your DAB develop using a microscope.

>*       wash, dehydrate, coverslip with DPX
>I have tried the Serotec product as well as the BD IHC product for Gr-1, 
>and various dilutions of primary to no avail.  Note that I get excellent 
>T-cell staining with CD4 and CD8a with this same protocol, with similar 
>distribution to the white pulp.  I have not found many examples of Gr-1 
>IHC in the literature.  Has anyone performed this staining routinely and 
>what type of distribution do you see?  Any comments or suggestions would 
>be greatly appreciated.

There is a picture of GR-1 on the BD website or should be.  You can contact 
their tech services about this also. You said nothing about your negative 
control, and it should be Rat IgG 2b isotype at the same concentration.

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

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