CD11b or CD68 for mouse brain, please

[Clear]

Hi all

I have no luck so far to make my immunoflouresence staining work for CD11b (from abcam) or CD68 (serotic),
 any suggestions? I hope that any body have got nice staining of these markers on mouse brain using   immunoflouresence technique to provide me the Cat num if possible. the mice brains that I am staining are 4%PFA fixed many thanks for the help in advance

[CanuckPhD]

Are your tissues in paraffin or frozen? If PFA fixed, sucrose protected and frozen here are the antibodies that I have had good luck with for microglia/macrophages.

Table 1: Antibodies used for immunohistochemistry
Antigen        (catalogue number)    Antibody type    Dilution    Source
CD16/CD32    (553140)                  Rat monoclonal   1:2000    BD PharMingen (San Jose, CA)
CD11b           (MCA711)                Rat monoclonal   1:250       Serotec (Raleigh, NC)
CD45             (MCA 1031G)            Rat monoclonal   1:5000     Serotec
CD68             (MCA 1957)              Rat monoclonal     1:500    Serotec
CD204          (MCA 1322)                Rat monoclonal     1:1000    Serotec
F4/80           (MCA497R)                Rat monoclonal      1:250     Serotec
MHC II           (553621)                  rat monoclonal      1:500     BD PharMingen

[dpajek]

@chanuckPhD: Do you know if these antibodies also work in the same dilution when using paraffine sections? Which antigen retrieval method would you suggest?
I hope you can help me with this as I am planning to use it in the near future. Thanks

[CanuckPhD]

Currently we usually use Iba-1 at 0.0005ug/ml (1:1000 dilution) following antigen retrieval using ph6.0 citrate buffer and polymer detection using a Leica IHC stainer. This is for mouse and rat.


I have also used the following in mouse paraffin 10um thick sections following antigen retrieval with citrate buffer pH6.0 and a standard ABC kit
MHC class II, 0.2ug/ml BD PharMingen;
CD45, 0.001 ug/ml, Serotec (this antigen does work better with formic acid antigen retrieval)

I found much better staining with frozen tissue as compared to paraffin for CD11b, F4/80 and CD16/32, therefore I usually stuck with frozen for these antigens

[CanuckPhD]

Sorry I must make a correction. The dilution for Iba-1 is 0.5ug/ml or 1:1000 of our stock solution. I forgot to convert to mg/ml first before I made the calculation (and I was too sure of myself to check our protocol book).

Sorry about that, Tim