Microglial Marker

Anti Iba1 Antibodies

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Iba1 (Ionized calcium-binding adapter molecule1) is used as a microglial marker because it is expressed specifically in microglia in the central nervous system. In peripheral tissues, it is expressed in macrophages and is also known as AIF-1. Fujifilm Wako's "Anti Iba1, Rabbit (for immunocytochemistry)" (Product Number 019-19741) is used by researchers all over the world as a standard for microglia marker antibody. In addition, Fujifilm Wako has also developed "Anti Iba1, Rabbit Monoclonal Antibody (6A4), recombinant" (Product Number 018-28523), which has the same performance as Anti Iba1, Rabbit (for immunocytochemistry).

About Iba1

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).

Anti Iba1 Antibodies of Fujifilm Wako

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..

01213_img14.png
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.

  • 01213_img15R3.png
    Trend in the annual number of publications
    Searched in Google scholar using the keyword "Iba1 019-19741 Wako"
  • Journal Number of Publications Impact Factor in 2022
    Nature 39 49.962
    Cell 35 41.582
    Science 2 47.728
    Nature Medicine 16 53.44
    Nature Neuroscience 65 24.884
    Nature Immunology 11 25.606
    Nature Biotechnology 7 54.908
    Nature Methods 4 28.547
    Nature Biomedical Engineering 5 25.671
    Nature Cell Biology 2 12.11
    Neuron 44 10.37
    Total 230  
01213_img28.png
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).

Product Lineup

Selection Flowchart

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*1 Experience with humans4) dogs5), cats6), pigs7), marmosets8), zebrafish9), etc. has been reported.
*2 Abbreviations:IHC(F)=Immunohistochemistry (frozen section), IHC(P)=Immunohistochemistry (paraffin section), ICC=Immunocytochemistry; WB=Western blotting

Product Lineup

Host Rabbit Goat Mouse
Product Number 018-28523 019-19741 013-27691 016-20001 016-26461 015-28011 012-28401 013-26471 011-27991 016-26721 013-27593
Description Rabbit monoclonal antibody For Immno- staining (Standard) For paraffin section For western blotting Biotin-conjugated SPICA Dye™ 568-conjugated SPICA Dye™ 594-conjugated Red fluorochrome (635)-conjugated Goat polyclonal antibody Mouse monoclonal antibody (NCNP24) Mouse monoclonal antibody (NCNP27)
Conjugate Unconjugated Unconjugated Unconjugated Unconjugated Biotin SPICA Dye™ 568
Ex=556 nm
Em=591 nm
SPICA Dye™ 594
Ex=575 nm
Em=611 nm
Red fluorochrome (635)
Ex=634 nm
Em=654 nm
Unconjugated Unconjugated Unconjugated
Antibody Monoclonal antibody Polyclonal antibody Monoclonal antibody
Concentration (mg/mL) 1.0-1.2 0.5-0.7 0.5-0.7 0.5-0.7 0.5-0.6 0.5-0.6 0.5-0.6 0.5-0.6 0.6-0.7 0.9-1.6 0.9-1.3
Antigen Synthetic peptide (Iba1 C-terminal sequence)
Cross-reactivity Mouse
Rat
Mouse
Rat
Other species also reported*1
Mouse
Rat
Human
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Mouse
Rat
Marmoset
Mouse
Rat
Human
Application*2 Figures indicate recommended concentrations IHC(F)
1:200-10,000
IHC(F)
1:500-1,000
ICC
1:500-1,000
IHC(P)
1:500-1,000
WB
1:500-1,000
IHC(F)
1:200-2,000
IHC(F)
1:200-2,000
IHC(F)
1:200-2,000
IHC(F)
1:200-2,000
IHC(F)
1:250-1,000
IHC(P)
1:250-1,000 WB 1:1,000
IHC(F, DAB)
1:500-2,000
IHC(F, fluorescent)
1:100
IHC(P, DAB)
1:100-1,000
Volume 100 μL 50 μg 50 μg 50 μg 100 μL 100 μL 100 μL 100 μL 100 μL 50 μL 50 μL
*1 Experience with humans4) dogs5), cats6), pigs7), marmosets8), zebrafish9), etc. has been reported.
*2 Abbreviations:IHC(F)=Immunohistochemistry (frozen section), IHC(P)=Immunohistochemistry (paraffin section), ICC=Immunocytochemistry; WB=Western blotting

Standard Protocol for Immunohistochemistry of Microglia using Iba1 antibody

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.

[Point] Samples without perfusion fixation or with inadequate fixation result in poor staining. Perfusion fixation with 4% paraformaldehyde-phosphate buffer is recommended.

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

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

[Point] Recommended thickness of tissue sections is 20-50 µm.

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.

[Point] 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

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.

[Point] 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.

3-2. Incubate overnight at 4°C.

[Point] Although it depends on the sample, rat cerebellum has been successfully stained with just 2 hours of incubation.

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.

[Point] 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.

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

[Point] If the background is high, shorten the incubation time for the secondary antibody. Recommended incubation time is 1-2 hours.

6.Washing

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

[Point] If the background is high, increase the number of washes.

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.

Application Data

Immunohistochemistry

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

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

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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)

01213_img17.png

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

01213_img18.png

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

Anti Iba1, Rabbit (for paraffin section)

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

Anti Iba1, Rabbit, Biotin-conjugated

01213_img20.png

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

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

Anti Iba1, Rabbit, SPICA Dye™ 594-conjugated

01213_img22.png

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

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

01213_img23.png

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

01213_img24.png

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)

01213_img25.png

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)

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 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

01213_img27.png

 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

References

  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

Product List

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Anti Iba1, Rabbit (Monoclonal Antibody)

Anti Iba1, Goat (Polyclonal Antibody)

Anti Iba1, Mouse (Monoclonal Antibody)

Anti Iba1, Mouse (Monoclonal Antibody)

For research use or further manufacturing use only. Not for use in diagnostic procedures.

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