[Histonet] reply to murine CD4 and CD8 in brain sections

From:Gayle Callis

Christophe Meiers wrote:

    Could  someone  please  give  advice regarding cd4 and cd8 staining of
    brain   tissue.    We  dissected  out  the  brain  of  a  mouse,  made
    longitudinal  section  midbrain,  one  half of the brain was placed in
    formalin for routine H & E, the other half was snap frozen for the cd4
    and  cd8.   The sections came out absolutely horrible with what looked
    like  exploded  tissue.  Should  the  brain  be treated in a different
    fashion ?  Would formalin interfere with staining CD4 and CD8 ?

*****You will have to frozen sections on fresh, unfixed snap frozen brain. 
Brain should be embedded in OCT, then it must MUST be snap frozen at very 
cold temps with either a dry ice/2 methylbutane mixture or another adequate 
method.  Snap freezing has been discussed many times on Histonet, for 
methods do a search of Histonet Archives at www.histosearch.org.   Formalin 
totally kills murine CD4 and CD8 antigen, and no retrieval or enzyme 
digestion will ever recover the antigens. Make sure block temperature and 
knife temperature are the same, equilibrate block (if stored at -80C) 20 
min or so before cutting.  Too cold a block means crunchy sections.

Cryosection brain at -16C, 5 um sections, pick up on Plus Charge slides, 
air dry overnight, fix in 75%acetone/25% absolute ethanol (no substitute on 
alcohol can be used) for 5 min at room temperature, go directly to a buffer 
rinse FROM the fixative, 3 changes of buffer, and proceed with staining. Do 
not store your frozen sections in cryostat after cutting, go directly to 
air drying.  We put our sections over a 16 mesh silica gel to ensure 
dryness.   Some people use acetone fixation at 4C for 10 min, but the 
acetone/alcohol improves morphology and keeps excellent immunostaining.

Peroxidase block, DAKO S2001 for 10 min

Normal serum block for 30 min (10% goat/2.5%mouse in TBS or Dulbeccos PBS 
with 0.05% Tween 20.  We add 0.2% goat serum to rinse buffers along with 
0.05% Tween 20

Avidin/biotin block kit Vector

CD4 Rat antimouse BD Pharmigen 1:500 (0.5mg/ml conc) in 5% goat serum.
CD8 Rat antimouse 1:200 (0.5mg/ml conc) in 5% goat serum
IgG isotype matched controls for these antibodies at same concentration in 
ug/ml is negative control
Incubate 30 min, RT

Goat antiRat F(ab')2 frag of IgG (0.5mg.ml) diluted 1:250 in normal serum 
block (yes! with mouse serum) for 30 min

Strepavidin-HRP 1:500 from Biosource/TAGO diluted in rinse buffer, 20 min

AEC+ DAKO control color development of positive control (normal spleen 
works) for approx 3 min

Rinse, counterstain, and mount with aqueous mounting media.

Gayle Callis
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)

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