[Histonet] Murine NALT and CD markers

[Gayle Callis]

Dear Lindsay,

You will not be able to fix OR decalcify NALT (nasal associated lymphoid 
tissue) from a mouse and have successful immunostaining.  Two options are 
remove NALT carefully, and do frozen sections - something we have done many 
times, OR cryosection nasal turbinates in undecalcified state, using 
CRYOJANE, from Instrumedics.

Murine CD4 and CD8 will never work with FFPE nor decalcification, 
decalcification does NOT work well after zinc TRIS buffer fixation as it 
will cause excessive swelling of tissue.  Fresh tissue frozen sections are 
best.

Removing NALT is not difficult. I can provide more details on this if you 
need it - privately.  Since NALT is on both sides of nasal passages, you 
have two that will be there when you dissect these out.  We snap freeze 
inside a very small Tissue Tek plastic mold  (10 x 10 mm) with NALT on 
bottom of mold, and palate side facing up.  Palate tissue is mostly 
connective tissue and tends to curl after removal so a tiny, thin layer of 
OCT in bottom of mold, then embed NALT in thin OCTlayer to flatten it out, 
add more OCT and quickly freeze by placing mold into a petri dish floating 
in liquid nitrogen.  DO NOT let liquid nitrogen get into petri dish, and 
support the dish to keep it from tipping over.  You will have a very flat 
face on block, and with care can obtain 50 or more serial sections of NALT.

Air dry frozens overnight at RT.  We have great success with murine CD 
markers, particularly with NALT and improved morphology using 75% 
acetone/25% absolute ethanol mixture to fix dried  FS for 5 min at RT but 
go directly to buffer after fixation, do not air dry again.

You wrote:

I want to look for mouse cell antigens in the NALT from decalcified
mouse heads, I know most antibodies to mouse cell surface markers do not
work in FFPE tissues. Any suggestions on how to fix the mouse heads and
decalcify and still be able to stain for CD4, CD8 etc...?
How about Zinc fixation with EDTA decalcification - or will the Zn ions
be chelated?
Lindsay


Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)