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Iba1 Antibody Staining Protocol for Immunohistochemistry
Description: Allograft inflammatory factor-1 (AIF-1 or Iba1) is a 17kDa cytoplasmic, cytokine-induced, calcium-binding protein first discovered in rat cardiac allografts with chronic rejection. AIF-1 is expressed in various human immunerelated tissues and may play a role in macrophage activation and function. AIF-1 is a marker of human microglia and is expressed by macrophages in injured skeletal muscle and regulates reduced proliferation and differentiation of satellite cells.
Primary Antibody
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Name: Iba1 Antibody |
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Clone: Mouse anti-Human |
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Supplier: Santa Cruz Biotechnology |
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Catalog Number: sc-32725 |
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Dilution: 1:200 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. For free floating sections: 4% paraformaldehyde fixed free floating sections. Primary antibody dilution 1:1000 overnight at 4 °C. |
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Incubation Time/Temp: 60 minutes/room temperature |
Antigen Retrieval
| Device: Steamer |
| Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) |
| Heat/Cool Temperature: 95 ºC - 100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Cytoplasmic of microglia and macrophages |
| Images: Search image |
Additional Information:
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Tissue Type: Brain |
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Fixation: Formalin fixed paraffin sections |
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Positive Control: Brain |
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Negative Control: Omit primary antibody, isotype control or absorption control |
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Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
Notes:
Not tested on frozen sections. |
References:
1. Daisuke Ito, MD; Kortaro Tanaka, MD; Shigeaki Suzuki, MD; Tomohisa Dembo, MD; Yasuo Fukuuchi, MD (2001) Enhanced Expression of Iba1, Ionized Calcium-Binding Adapter Molecule 1, After Transient Focal Cerebral Ischemia In Rat Brain. Stroke. 32:1208.
2. Richard P. Kennan, Fabiana S. Machado, Sunhee C. Lee, Mahalia S. Desruisseaux, Murray Wittner, Moriya Tsuji and Herbert B. Tanowitz (2005) Reduced cerebral blood flow and N-acetyl aspartate in a murine model of cerebral malaria Parasitol Res. 96(5):302-7.
3. Jörgen Henriksson and Hans Tjälve (2000) Manganese Taken Up into the CNS via the Olfactory Pathway in Rats Affects Astrocytes. Toxicological Sciences 55:392-398