Q: What is the antigen?

A: It is a synthetic peptide of Iba1 (homologous to the C-terminal sequence). The specific sequence is not disclosed.

Q: How many times can 50 µg (1 vial) be used?

A: For immunohistochemistry, when 200 μL of antibody solution at a 1:1000 dilution is used per slide, the antibody can be used approximately 500 times.

Q: What secondary antibodies can be used?

A: At FUJIFILM Wako, the following secondary antibodies have been successfully used:

Alexa Fluor® 488-AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, Product Number 111-545-144）
Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H+L)　(Jackson ImmunoResearch, Product Number 711-605-152)

Q: No band is detected by Western blotting.

A: This antibody is suitable for immunohistochemistry (frozen sections) and immunocytochemistry. For Western blotting, use Anti Iba1, Rabbit (for Western blotting) (Product Number 016-20001).

Q: Microglia are not stained by immunohistochemistry or immunocytochemistry.

A: The causes below are possible. Try the solutions listed.

1. Inadequate fixation
   It has been reported that samples without perfusion fixation or with inadequate fixation show reduced staining. For immunohistochemistry, prepare frozen sections after perfusion fixation with 4% paraformaldehyde.
2. The antigen is denatured.
   Try antigen retrieval under the following conditions:
   (A) Citrate buffer (pH 6.0) at 90℃ for 9 minutes
   (B) TE buffer (pH 9.0) at 90℃ for 9 minutes
3. Samples are deteriorating.
   New samples should be prepared. Thickness of the sections should be 20-50 µm.
4. Insufficient antibody concentration
   Use a higher concentration of the antibody. Recommended dilution is 1:500-1:1000.

Q: The background of immunohistochemistry and immunocytochemistry is high.

A: The causes below are possible. Try the solutions listed.

1. Inadequate blocking
   Try extending the incubation time for blocking or use a different blocking agent. In addition to PBS containing 1% BSA and 0.3% TritonX-100 as described in the recommended protocol in the instruction manual, PBS containing 1% BSA + 0.3% Tween-20, and 3% normal serum from the host of the secondary antibody are also recommended.
2. Excessive primary antibody
   Use a lower concentration of the primary antibody. The recommended dilution of the anti Iba1 antibody is 1:500-1:1000.
3. Excessive reaction time for the secondary antibody
   Shorten the reaction time. The recommended reaction time is 1-2 hours. Alternatively, increase the number of washings after the reaction with the secondary antibody.
4. Samples are deteriorating.
   Samples should be prepared again. Thickness of the sections should be 20-50 µm.
5. The antigen is denatured.
   Try antigen retrieval under the following conditions:
   (A) Citrate buffer (pH 6.0) at 90℃ for 9 minutes
   (B) TE buffer (pH 9.0) at 90℃ for 9 minutes
6. Endogenous peroxidases are reacting (if HRP or POD is used as a detection enzyme)
   To inactivate the endogenous peroxidases, treat the specimen with 80% methanol containing 3% hydrogen peroxide at -20℃ for 20 minutes before blocking.

Q: In immunohistochemistry, neurons are stained in addition to microglia.

A: The causes below are possible. Try the solutions listed.

1. Excessive antibodies
   Use a lower concentration of primary or secondary antibody. The recommended dilution of anti Iba1 antibody is 1:500-1:1000.
2. Excessive reaction time for the secondary antibody
   Shorten the reaction time. The recommended reaction time is 1-2 hours. Alternatively, increase the number of washings after the reaction with the secondary antibody.
3. The antigen is denatured.
   Try antigen retrieval under the following conditions:
   (A) Citrate buffer (pH 6.0) at 90℃ for 9 minutes
   (B) TE buffer (pH 9.0) at 90℃ for 9 minutes

Q: Can this antibody be used for flow cytometry?

A: Although we do not have any data on this use, this antibody has been used in the following study:

Koh, H. S., et al.: Nat. Commun., 6(1),1(2015).
The HIF-1/glial TIM-3 axis controls inflammation-associated brain damage under hypoxia.