I tried Iba-1 (WAKO) to stain microglia from frozen human brain biopsy tissue but it didnt work. Frozen brain biopsy tissue (1-2 mm) was fixed in 4% PFA for 2 hrs, sucrose impregnated, stored in antifreeze. 35 micron sections were cut on cryostat .I did use 0,3% H2O2 in methanol pretreatment followed by blocking with 10% normal goat serum. After overnight incubation in primary and 2 hr incubation with secondary, color was developed with NiDAB. Can anyone help me with troubleshooting tips?
Your conditions sound fine- although you have not included the primary concentration. Do you have a lot of experience with IHC, and specifically have you got other IHC working in these tissue. Attached is a recent paper using Iba-1 in human tissue - and the Wako antibody is something we have also used in this lab for human microglia- using a very similar set of conditions to what you have used.
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Thanks Bobbi, I used 1:5000 primary for free floating sections. I have experience with rodent tissue, actually this is first try with human tissue.
I would try a titration. It has been used in perintal tissues at 1:1000, so i would suggest a 1:500, 1:1000 and and 1:2000 trial of the primary antibody. I would keep your secondary constant at 1:200 - and once you have a signal you could possible try and reduce any background by further titration of the secondary. This paper (Monier 2006) has very detailed methods of staining human microglia, including dilutions of antibodies. For the titrations use adjacent sections if possible, but sections that might otherwise be damaged or not best for your main study. Good luck!
Article Distribution and differentiation of microGlia in the human e...
Attached is a recent paper using Iba-1 in human samples and the Wako antibody. They recommend 1:2000 dilution.
In rat tissue I have always used 1:300 dilution with this same antibody. I agree with Bobbi, once you have signals you can play with dilutions.
We have tried CD45 and F4/80 on human autopsy AD brain sections and they seem to work fine in recognizing microglia/macrophages. If you have access to these antibodies, please try them as well. Also, it would be good idea to include rat/mouse brain sections when you process human brain sections, just to make sure that the antibodies are good and indeed they work as expected.
Thanks Kanchan, I will play with various dilutions. Kiran, we do not know for sure if these biopsy tissues are positive for amyloid plaques. we took Iba-1 which is good marker for both angry as well as placid microglia.
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