First of all, are the cell types there, you need to have a good postive
control particularly for the macrophage i.e. inflammed skin?? Normal
spleen for others.
Frozen sections, air dried overnight, 4C acetone 10 min, or acetone/alcohol
fixation is superb - 75 mls acetone, 25 mls alcohol- air dry frozens
overnight, fix 5 min room temp, then go to 3 rinses PBS immediately.
Glucose oxidase peroxidase block, methanolic peroxide blocking is not
recommended for CD markers, you can try peroxide in PBS to avoid the
Normal serum block 10% goat/2.5% mouse 30 min for rat antimouse monoclonal
primaries, use 10% goat with CD3.
Avidin/biotin block Vector or other kit
do a dilution panel for all antibodies, target concentration to start with
10 ug/ml - work on either side of that, CD11b and CD45R will stain
concrete! CD3 (ours is an Armenian hamster clone so secondary is goat
antiArmenian Hamster-biotin adsorbed to mouse, diluted 1:250 from Jackson
Immunoresearch) or Pharmingen hamster cocktail. Incubation 30 min, RT
With B220 and MAC1, the secondary is always goat antiRat F(ab')2 fragment
of IgG, from Biosource/TAGO immunologicals, 0.5mg/ml diluted 1:250 for 30
min RT. This F(ab')2 secondary prevents binding to fc receptors on tissue,
particularly with close species. This secondary is also diluted in the
NORMAL SERUM BLOCK.
Strepavidin-HRP, your choice 20 min RT,
Chromogen is AEC+ from DAKO
If you try paraffin sections - try enzyme digestion, but your primary
antibodies will be more concentrated than what is used on frozen sections,
and you might have to incubate overnight at 4C.
There is a formalin free Zinc TRIS Buffer fixative in Pharmingen
catalog/website for paraffin sections that gives ok morphology, and
excellent CD marker staining.
B220 I have unsuccessfully tried staining frozen mouse skin tissue for
immune markers such as cd45r (b cell), cd3(t cell), cd11b (macrophage).
Can anyone offer suggestions regarding the use of appropriate antibodies (I
have been using antibodies from Pharmingen), fixation, antigen retrieval,
blocking, or anything else that might help in staining for the above cell
types in mouse skin tissue.
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610
406 994-4303 (FAX)
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