[Histonet] Re: Mouse CD45 staining of bone marrow

[Johnson, Teri]

Dear Adam,

Thank you for your detailed email regarding your problem with CD45 immunostaining! That helps tremendously know where to start in a reply. I have cc'd the Frozen section CD marker guru on this, so expect an equally detailed response to this in the near future. She'll be able to tell you how to proceed here, but I suspect your problem is aldehyde fixation. The amount of time the cells are fixed for flow cytometry is very minimal. Quite different from the overnight treatment your protocol uses.

Gayle will likely tell you to use unfixed, undecalcified Cryojane sectioned bone sections that are post-sectioning fixed in Acetone/Ethanol 3:1.

First I need to know if the immunostaining protocol you are using is currently providing nice strong immunostaining for other markers. In other words, is the detection you are using a sound one at those conditions? If no, then optimize your detection system first. If yes, then read on.

If you are liking the morphology of the fixed and cryopreserved bone (and who wouldn't?) then you can perhaps try using Zinc Formalin as a fixative prior to sucrose cryoprotection and see if that works.

Some CD markers work only with Zinc Tris buffer (Beckstead), that contains no aldehyde. You can find tons of information on this by doing a histonet or google search. Fix the tissues with the Zinc Tris, decalcify in EDTA, section, and then do what you usually do.

For the samples you already have available, maybe try using some gentle HIER with citrate pH 6.0 or a higher pH, 60 degrees C for 10-20 minutes to see if you can recover some of the antigens. Yes, you can do AR on frozen sections on fixed tissues.

Ok, start there. This is just a mere appetizer compared to the full course meal you are about to get.

Enjoy, and good luck!

~Teri

Teri Johnson, HT(ASCP)QIHC
Head, Histology and Electron Microscopy
Stowers Institute for Medical Research
Kansas City, MO