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Iba1 Antibody Staining Protocol for Immunohistochemistry
Description: 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 cells.
Primary Antibody
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Name: Iba1 (ionized calcium binding adapter molecule 1) Antibody |
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Clone: Rabbit polyclonal |
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Supplier: Wako Pure Chemical Industries, Ltd. |
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Catalog Number: 019-19741 |
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Dilution: 1:500 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: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102) |
| 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. Specific to microglia and macrophages, but not cross-reactive with neurons and astrocytes. |
| 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