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

Dear Collin Gagne ,

You may try this:

"Hippocampal sections were equilibrated at RT, washed with PBS and blocked with 10% bovine serum albumin in 0.25%TritonX-100/PBS (PBS-T) for 1 hour at RT. Thereafter, sections were incubated with primary antibody (anti-Iba-1, Wako, 1:500 diluition; Cat# 019-19741; RRID:AB_839504) diluited in PBS-T overnight at RT. Sections were rinsed with PBS, and incubated with Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (1:2,000 diluition, Molecular Probes) diluited in PBS-T for 2h at RT."

Melo et al., 2020, Cell Reports 30, 2180–2194

https://doi.org/10.1016/j.celrep.2020.01.072