"Anti Iba1, Rabbit (for Immunocytochemistry)" is an excellent microglial marker antibody since it can stain even microglial processes. Here, we describes the protocol and points to note when performing microglial immunohistochemistry. Frozen sections of mouse brain and a fluorescent dye are used as an example.

1. Mice are perfused and fixed in 4% paraformaldehyde-phosphate buffer.
   1. Note: Samples without perfusion fixation or with inadequate fixation result in poor staining. Perfusion fixation with 4% paraformaldehyde-phosphate buffer is recommended.
2. Replace with sucrose, and prepare frozen blocks.
3. Prepare 50 µm-sections in thickness using a microtome.
   1. Note: Recommended thickness of tissue sections is 20-50 µm.

1. Wash with 0.3% TritonX-100/PBS 3 times for 5 minutes each.
2. Block in 1% BSA, 0.3% TritonX-100/PBS for 2 hours at room temperature.
   1. Note: If background is high, try extending the incubation time for blocking or changing the blocking solutions. The following blocking solutions can also be used:

• • • 1% BSA, 0.3% Tween-20/PBS
    • 3% normal serum of the host of the secondary antibody

1. Add Anti Iba1, Rabbit (for immunohistochemistry) to 1% BSA, 0.3% TritonX-100/PBS at 1:1,000 dilution.
   1. Note: If the staining is weak, increase the antibody concentration. If the background is high, decrease the antibody concentration. Recommended dilution is 1:500-1,000.
2. Incubate overnight at 4°C.
   1. Note: Although it depends on the sample, rat cerebellum has been successfully stained with just 2 hours of incubation.

1. Wash with 0.3% TritonX-100/PBS 3 times for 5 minutes each.

1. Add fluorescent-labeled anti-rabbit IgG antibody (e.g. Jackson ImmunoResearch, Product No. 111-545-144), to 1% BSA, 0.3% TritonX-100/PBS at 1:1,000 dilution.
   1. Note: If the staining is weak, increase the antibody concentration. If the background is high, decrease the antibody concentration. Recommended dilution is 1:500-1,000.
2. Incubate for 2 hours at room temperature.
   1. Note: If the background is high, shorten the incubation time for the secondary antibody. Recommended incubation time is 1-2 hours.

1. Wash with 0.3% TritonX-100/PBS 3 times for 5 minutes each.
   1. Note: If the background is high, increase the number of washes.

1. Mount the sections in mounting media.

1. Observe the sections under a fluorescence microscope or a confocal microscope.

If microglia do not stain well using the techniques described at left, perform antigen retrieval after sectioning using one of the following:

1. Citrate buffer (pH 6.0) for 9 minutes at 90°C
2. TE bufferi (pH 9.0) for 9 minutes at 90°C

Other troubleshooting is also available in the FAQ.