Anti Iba1, Rabbit (for Immunocytochemistry)
- for Immunochemistry
- Manufacturer :
- FUJIFILM Wako Pure Chemical Corporation
- Storage Condition :
- Keep at -20 degrees C.
- Source :
- Rat
- Cross‒Reactivity :
- Human, Mouse, Rat
- Host :
- Rabbit
- Application :
- ICC, IHC(Frozen)
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- Packing
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50ug
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In stock in Japan |
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Product Overview
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 microglia marker antibody standard.
Features
- Enables even the processes of microglia to be immunohistochemical-stained*.
- Relatively small amount needed, with concentrations ranging from 1:500 to 1:1,000.
* The result of staining depends on sample condition. Staining performance is not guaranteed.
Antibody Information
| Clonality | Polyclonal |
|---|---|
| Antigen | Synthetic peptide (Iba1 C-terminal sequence) |
| Host | Rabbit |
| Reactivity* | Mouse, Rat |
| Concentration (mg/mL) | 0.5 - 0.7 |
| Applications | Immunohistochemistry (Frozen Section) 1:500 - 1,000 Immunocytochemistry 1:500 - 1,000 |
* Experience with humans4) dogs5), cats6), pigs7), marmosets8), zebrafish9), etc. has been reported.
Standard Protocol for Immunohistochemistry of Microglia using Iba1 antibody
"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.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.
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.
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. .
Application Data
Immunohistochemistry (Mouse)
| Species | Mouse |
| Site | Cerebellum |
| Sample | Frozen section |
| Antibody concentration | 1:1,000 |
| Species | Mouse |
| Site | Nucleus accumbens core |
| Sample | Slice section |
| Antibody concentration | 1:500 |
| Data by courtesy of | Dr. Hagiwara, Mr. Yamazaki Tokyo University of Science |
| Species | Mouse |
| Site | Spinal cord |
| Sample | Frozen section |
| Antibody concentration | 1:500 |
| Data by courtesy of | Dr. Hagiwara, Mr. Sawada Tokyo University of Science |
| Species | Mouse |
| Site | Retinal whole mount |
| Antibody concentration | 1:1,000 |
| Data by courtesy of | Prof. Lieve Moons KU Leuven Belgium |
Immunohistochemistry (Rat)
| 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 |
References
- 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 - 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 - 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. - Zhao, S. et al.: Cell, 180(4), 796(2020).
Cellular and Molecular Probing of Intact Human Organs - 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 - Ide, T. et al.: J. Vet. Med .Sci., 72(1), 99 (2010).
Histiocytic Sarcoma in the Brain of a Cat - 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 - Rodriguez-Callejas, J.D. et al.: Front. Aging Neurosci., 8, 315(2016).
Evidence of Tau Hyperphosphorylation and Dystrophic Microglia in the Common Marmoset - 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
FAQ
About antibody
- What is the antigen?
- It is a synthetic peptide of Iba1 (homologous to the C-terminal sequence). The specific sequence is not disclosed.
- How many times can 50 µg (1 vial) be used?
- 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.
About protocol
- What secondary antibodies can be used?
- 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)
About trouble shooting
- No band is detected by Western blotting.
- This antibody is suitable for immunohistochemistry (frozen sections) and immunocytochemistry. For Western blotting, use Anti Iba1, Rabbit (for Western blotting) (Product Number 016-20001).
- Microglia are not stained by immunohistochemistry or immunocytochemistry.
- 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.
- The background of immunohistochemistry and immunocytochemistry is high.
- 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.
- 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
About application
- Can this antibody be used for flow cytometry?
- 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.
Overview / Applications
| Outline | This product is for research use only. Do not administer it to human. Calcium ions are known to be one of the most important signal mediators in all cells including central nervous system (CNS) cells. Calcium ions exert their signaling activity through association with various calcium binding proteins, many of which are classified into a large protein family, the EF hand protein family.Iba1 is a 17-kDa EF hand protein that is specifically expressed in macrophages/ microglia and is upregulated during the activation of these cell. Wako has launched rabbit polyclonal antibodies were raised against a synthetic peptide corresponding to the Iba1 carboxy-terminal sequence, which was conserved among human, rat and mouse Iba1 protein sequences. Rabbit Anti Iba1 antibody is raised a synthetic peptide corresponding to C-terminus of Iba1. Purified by the antigen affinity chromatography from rabbit antisera and prepared in TBS solution. Contains no preservatives and stabilizers. Reactive with mouse and rat Iba1. Working Concentration: Immunocytochemistry 1-2ug/mL Immunohistotochemistry 0.5-1ug/mL Storage: Store at -20C. After opening, aliquot contents and freeze at -20C. Package Size:50ug (100uL) References:Imai, Y., Ibata, I., Ito, D., Ohsawa, K. and Kohsaka, S.: Biochem. Biophys. Res. Commun., ;224, 855 (1996). Ito, D., Imai, Y., Ohsawa, K, Nakajima, K, Fukuuchi, Y. and Kohsaka, S.: Brain Res. Mol. Brain Res., 57, 1 (1998). Ohsawa, K., Imai, Y., Kanazawa, H., Sasaki, Y. and Kohsaka, S.: J. Cell Sci., 113, 3073 (2000). Sasaki, Y., Ohsawa, K., Kanazawa, H., Kohsaka, S. and Imai, Y.: Biochem. Biophys. Res. Commun., 286, 292 (2001). Kanazawa, H., Ohsawa, K., Sasaki, Y., Kohsaka, S. and Imai, Y.: J. Biol. Chem.,. 277, 20026(2002). |
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Antibody Information
| Antigen | Iba1 |
|---|---|
| Antigen Alias | Iba1 Iba 1 |
Manufacturer Information
Alias
For research use or further manufacturing use only. Not for use in diagnostic procedures.
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