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

Name: Iba1 Antibody

Clone: Mouse anti-Human

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-32725

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Cytoplasmic of microglia and macrophages
Images: Search image

Additional Information:

Tissue Type: Brain

Fixation: Formalin fixed paraffin sections

Positive Control: Brain

Negative Control: Omit primary antibody, isotype control or absorption control

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


Not tested on frozen sections.



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