Sonya,
I guess my very greatest concern is from your first sentence.  You did flow but "didn't see anything".  I guess I would never even attempt IHC if I didn't see target verified by flow for an uncharacterized target.  Mouse spleenocytes stained with 33D1 do give a one log shift and there are enough of them you can see them.  This is not a shoulder off of the negative peak.  I've seen numbers of target sites per cell estimated at 10,000 molecules per cell so this should be seen.  Assuming you are not using a very strange strain of mouse since haplotype restriction has not been fully assessed.  As far as the BMDC's plated to glass after GMCSF stimulation.  Were these verified?  In-vivo stimulations sometimes do fail with resultant lack of expansion of the targeted population.  My cell culture experimental stimulations were never 100% successful.  No ones are due to multiple reasons.  After 5 days in GMCSF, were those cells flowed?  I think originally you said the coverslip staining was 
"weak" and that shouldn't be.  Check them by flow first.  Bad GMCSF, wrong dilution calculation?  Your stain setup looks good - it does and should work.  The markers CD11c and CD205 are fine and MIDC-8 is another.  But I guess my bottom line first and foremost is I wouldn't waste even another second doing IHC until I can stain 33D1 in flow and on those coverslips just to be sure my control standards are there and functioning.  If you are flowing mouse splenocytes, can pick up a dendritic cell population by CD11c or 205, and then can't see any 33D1 at all - something is wrong long before IHC problems are there.  Bad mouse, bad 33D1, bad Alexa 488, bad something but it is a lot easier to troubleshoot flow or coverslip staining of stimmed BMDC's and get that working than to troubleshoot IHC staining up front.
Ray

-------------- Original message -------------- 
From: "Martin S."  

Hi All,

Some further info;

The sections were mouse spleen - we did some flow analysis with 33D1 of splenocytes but didnt see anything - hard to know if this is because there are just so few 33D1+ cells or if theres a problem with the antibody. There are a few papers out there with quite a lot of staining with 33D1 in mouse spleen - most recently in Science (Dudziak et al Vol 315 Jan 2007). For frozen sections I have tried 33D1 + mouse anti-rat IgG2b:Biotin with Streptavidin Alexa488 and also 33D1 + rb anti-rat:Biotin with tyramide amplification and Streptavidin Alexa488.
The coverslip DC's were BMDC's - BM cells were harveted from mice and grown with GMCSF for 5 days before transfering cells to PLL coated coverslips. Cells were allowed to attach for 2hrs and were then fixed with 4% PFA, 10min.
Basically we want to look at DC's in the spleen in conjuction with a number of other cell markers. We are using CD11c and Dec205 antibodies but wanted an additional DC marker.

Thanks for all your suggestions/comments

Sonya
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