Anti Iba1 Antibodies

Iba1 (Ionized calcium-binding adapter molecule1) is an approximately 17 kDa calcium-binding protein. It is used as a microglial marker because it is expressed specifically in microglia in the central nervous system¹⁾. It is expressed in both resting and activated microglia, but is reportedly expressed more highly in activated microglia²⁾. It is also expressed in macrophages in peripheral tissues and is known as AIF-1 (Allograft inflammatory factor-1).

Iba1 binds to F-actin in cells to form actin bundles. The formation of actin bundles is thought to be required for the membrane ruffling observed during cell migration and phagocytosis³⁾.

Fujifilm Wako’s "Anti Iba1, Rabbit (for immunocytochemistry)" (Product Number 019-19741), which allows even microglia processes to be stained by immunohistochemical staining, is used by researchers all over the world as a standard for microglia marker antibody..

Immunohistochemical staining
of mouse cerebellum
Anti Iba1, Rabbit
(for immunocytochemistry)

Annual number of publications

Anti Iba1, Rabbit (for immunocytochemistry) has been appearing in an increasing number of publications, including many articles published in top journals (Nature, Cell, Neuron, etc.), and the annual number of such publications exceeded 1,300.

Reference List

Journal	Number of Publications	Impact Factor in 2023
Nature	44	69.5
Cell	38	45.5
Science	2	44.7
Nature Medicine	18	58.7
Nature Neuroscience	83	21.2
Nature Immunology	12	27.7
Nature Biotechnology	8	68.1
Nature Methods	4	51.3
Nature Biomedical Engineering	5	29.2
Nature Cell Biology	3	26.6
Neuron	52	16.2
Total	269

Immunohistochemical staining
of mouse cerebral cortex
Anti Iba1, Rabbit
Monoclonal Antibody (6A4), recombinant

Development of rabbit monoclonal antibody

In 2023, Fujifilm Wako launched a rabbit monoclonal antibody, "Anti Iba1, Rabbit Monoclonal Antibody (6A4), recombinant" (Product Number 018-28523). We have confirmed that this antibody has the same performance as “Anti Iba1, rabbit (for immunocytochemistry)” in immunohistological staining of mice and rat brains. It has also been reported to obtain good results in immunohistochemical staining of mouse retinas (see application data).

Selection Flowchart

Product Lineup

Fujifilm Wako's "Anti Iba1, Rabbit (for Immunocytochemistry)" (Product No. 019-19741) 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.Preparation of tissue sections

1-1. Mice are perfused and fixed in 4% paraformaldehyde-phosphate buffer.

1-2. Replace with sucrose, and prepare frozen blocks.

1-3. Prepare 50 µm-sections in thickness using a microtome.

2.Washing - Blocking

2-1. Wash with 0.3% TritonX-100/PBS 3 times for 5 minutes each.

2-2. Block in 1% BSA, 0.3% TritonX-100/PBS for 2 hours at room temperature.

3.Primary antibody reaction

3-1. Add Anti Iba1, Rabbit (for immunohistochemistry) to 1% BSA, 0.3% TritonX-100/PBS at 1:1,000 dilution.

3-2. Incubate overnight at 4°C.

4.Washing

4-1. Wash with 0.3% TritonX-100/PBS 3 times for 5 minutes each.

5.Secondary antibody reaction

5-1. 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.

5-2. Incubate for 2 hours at room temperature.

6.Washing

6-1. Wash with 0.3% TritonX-100/PBS 3 times for 5 minutes each.

7.Mounting

7-1. Mount the sections in mounting media.

8.Observation

8-1. 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:
(A) Citrate buffer (pH 6.0) for 9 minutes at 90°C
(B) TE bufferi (pH 9.0) for 9 minutes at 90°C

Other troubleshooting is also available in the FAQ on the product detail page.

Immunohistochemistry

▼Anti Iba1, Rabbit Monoclonal Antibody (6A4), recombinant

Species: Mouse
Site: Cerebral cortex
Sample: Frozen section
Antibody concentration: 1:1,000

Species: Mouse
Site: Retina
Sample: Frozen section
Antibody concentration: 1:2,000
Data by courtesy of
Dr. Watanabe, Dr. Iwagawa
University of Tokyo Hospital

▼Anti Iba1, Rabbit (for immunocytochemistry)

Species: Rat
Site: Cerebral cortex
Sample: Frozen section
Antibody concentration: 1:1,000
Data by courtesy of
Dr. Sanagi, Dr. Manabe,
Dr. Ichinohe, Dr. Kohsaka
National Center of
Neurology and Psychiatry

Species: Mouse
Site: Cerebellum
Sample: Frozen section
Antibody concentration: 1:1,000

Species

Mouse

Site

Cerebral cortex/Area postrema (Medulla oblongata)

Sample

Frozen section

Antibody concentration

P2RY12 → Anti P2RY12, Guinea Pig (Product No. 011-28873) 1:900
Iba1 → Anti Iba1, Rabbit (for Immunocytochemistry) 1:500
Laminin → Anti Laminin Antibody (Rat, Made in Dr. Miyata’s Lab) 1:200

Data by courtesy of
Dr. Miyata, Department of Applied Biology, Kyoto Institute of Technology

[Result]
Among the Iba1-positive cells, those that are present in the brain parenchyma are P2RY12-positive and are thought to be microglia (arrowheads). On the other hand, Iba1-positive/P2RY12-negative cells are present around the blood vessel basement membrane, and it is thought that these cells are macrophages (arrows).

▼Anti Iba1, Rabbit (for paraffin section)

Species: Rat
Site: Hippocampus vicinity
Sample: Paraffin section
Antibody concentration: 1:1,000

▼Anti Iba1, Rabbit, Biotin-conjugated

Species: Rat
Site: Cerebral cortex
Sample: Frozen section
Antibody concentration: 1:200
Data by courtesy of
Dr. Sanagi, Dr. Manabe,
Dr. Ichinohe, Dr. Kohsaka
National Center of
Neurology and Psychiatry

▼Anti Iba1, Rabbit, SPICA Dye™ 568-conjugated

Species: Rat
Site: Cerebral cortex
Sample: Frozen section
Antibody concentration: 1:200

▼Anti Iba1, Rabbit, SPICA Dye™ 594-conjugated

Species: Rat
Site: Cerebral cortex
Sample: Frozen section
Antibody concentration: 1:200

▼Anti Iba1, Rabbit, Red Fluorochrome (635)-conjugated

Species: Rat
Site: Cerebral cortex
Sample: Frozen section
Antibody concentration: 1:200
Data by courtesy of
Dr. Sanagi, Dr. Manabe,
Dr. Ichinohe, Dr. Kohsaka
National Center of
Neurology and Psychiatry

▼Anti Iba1, Goat

Species: Mouse
Site: Hippocampus
Antibody concentration: 1:200
Data by courtesy of
Dr. Takata
Division of Integrated
Pharmaceutical Sciences,
Kyoto Pharmaceutical University

▼Anti Iba1, Mouse Monoclonal Antibody (NCNP24)

Species: Rat
Site: Cerebral cortex
Sample: Frozen section
Antibody concentration: 1:1,000
Data by courtesy of
Dr. Sanagi, Dr. Manabe,
Dr. Ichinohe, Dr. Kohsaka
National Center of
Neurology and Psychiatry

Western blotting

▼Anti Iba1, Rabbit (for Western Blotting)

　1.Iba1 protein...10 ng
　2.Rat Microglia...10 μg
　3.Rat Neuron...10 μg
　4.Rat cerebral cortex...150 μg

SDS-PAGE: 5.5% stacking gel, 12.5% running gel, 100V
Blocking: 3% skim milk/TBS, 1 hour, room temperature.
Primary antibody: 1:1,000 concentration, 3% skim milk/TTBS, overnight, 4℃
Secondary antibody: Peroxidase-labeled Anti Rabbit IgG (1/5,000), 3% skim milk/TTBS, 1 hour, room temperature

▼Anti Iba1, Goat

　1.Rat primary culture microglia...10 μg
　2.Rat primary culture neuron...10 μg
　3.Rat primary culture astrocyte...10 μg
　4.Rate cerebral cortex...100 μg

Primary antibody: Anti Iba1, Goat, 1:1,000
Secondary antibody: Anti Goat IgG, HRP-conjugated

1. Imai, Y., Ibata, I., Ito, D., Ohsawa, K. & Kohsaka, S.: Biochemical and biophysical research communications, 224(3), 855(1996).
   A Novel Geneiba1 in the Major Histocompatibility Complex Class III Region Encoding an EF Hand Protein Expressed in a Monocytic Lineage
2. Mori, I., Imai, Y., Kohsaka, S. & Kimura, Y.: Microbiology and immunology, 44(8), 729(2000).
   Upregulated expression of Iba1 molecules in the central nervous system of mice in response to neurovirulent influenza A virus infection
3. Sasaki, Y., Ohsawa, K., Kanazawa, H., Kohsaka, S. & Imai, Y.: Biochemical and biophysical research communications, 286(2), 292(2001).
   Iba1 is an actin-cross-linking protein in macrophages/microglia.
4. Zhao, S. et al.: Cell, 180(4), 796(2020).
   Cellular and Molecular Probing of Intact Human Organs
5. Ahn, J.H. et al.: Lab. Anim. Res., 28(3), 165 (2012).
   Comparison of alpha-synuclein immunoreactivity in the spinal cord between the adult and aged beagle dog
6. Ide, T. et al.: J. Vet. Med .Sci., 72(1), 99 (2010).
   Histiocytic Sarcoma in the Brain of a Cat
7. Gaige, S. et al.: Neurotoxicology, 34, 135(2013).
   c-Fos immunoreactivity in the pig brain following deoxynivalenol intoxication: Focus on NUCB2/nesfatin-1 expressing neurons
8. Rodriguez-Callejas, J.D. et al.: Front. Aging Neurosci., 8, 315(2016).
   Evidence of Tau Hyperphosphorylation and Dystrophic Microglia in the Common Marmoset
9. Fantin, A. et al.: Blood, 116(5), 829(2010).
   Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction