[Histonet] Gr-1 staining in mouse spleen

[Herber, Donna L.]

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.
*	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. 
*	Wash 3x PBS 5 min
*	Permeabilize 30 min with 1.8% lysine. 0.2% triton X-100, 4% serum, in PBS
*	Quick rinse in PBS
*	Direct application of antibody BD# 553123 incubate overnight.
*	Wash 3x PBS 5 min
*	Vector biotinylated secondary #BA4001 rabbit anti-rat, 1:1000 dilution, 2 hours
*	Wash 3x PBS 5 min
*	Vectastain ABC kit PK-4000 one hour
*	Wash 2x PBS, 1x TBS,  5 min
*	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
*	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.


Donna Herber, Ph.D.
Post Doctoral Scholar
Immunology (Gabrilovich Laboratory)
H. Lee Moffitt Cancer Center and Research Institute
745-6097



Donna Herber, Ph.D.
Post Doctoral Scholar
Immunology (Gabrilovich Laboratory)
H. Lee Moffitt Cancer Center and Research Institute
745-6097


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