I've been trying various iba1 antibodies with either Alexa 488 or 647 and am getting a lot of background and have never once seen anything close to resembling a microglia. I've tried blocking with various combinations of 0, .3 or 3.0% milk, 0, .3 or 1.0% BSA, with 4% normal donkey serum and nothing has worked.
Any suggestions would be very much appreciated. Perhaps there are better membrane-bound proteins I could stain for?
I’ve had good experience with Iba-1 blocking in 10% goat or donkey serum, followed by 1% serum in the primary antibody solution.
Dear Collin Gagne ,
I do the same as Megan Duffy Peterson and I have really good results as well. Besides this, it’s very important to control the background by performing some steps such as blocking endogenous peroxidase activity with methanol and h2o2, and blocking endogenous biotin using avidin and then biotin.
Good Luck.
Marcus
Hello Collin Gagne ,
I have good results for Iba1 in 4%PFA-fixed tissue with antibody solution in PBS-1%BSA, after blocking incubation. The same for the secondary one. It is true that I have got better images with 488 than 647 or 594...
Wich Iba1 primary antibody do you use?
Best
Hi!
i usually block for 30 mins (but you can go up to 1 hour) with 4.5% NGS in PBS-T (triton 0.2%) at room temperature and in dark.
then also primary and secondary antibodies are diluted in 3% NGS in PBS-T (triton 0.2%).
as antibody i use the traditional IBA-1 from WAKO, either 1/1000 or 1/3000.
i also first suggest you to make a BF staining, to really see if there are some problems with your tissues and test the antibodies you have
cheers
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