Blocking/Dilution buffer: 5% NFDM/TBST.

IBA1 is a relatively minor protein of brain and is much more abundant in spleen (PMID: 8912632, PMID: 29232670). We suggest loading higher amount of brain lysate or using lower dilution of antibody for detecting signal in brain related lysates.

Slides were washed with 1x PBS and then blocked in 5% goat serum containing 0.3% Triton X-100. Iba-1 was stained using ab178847 at 1/500 dilution in immunohistochemical analysis. Representative immunofluorescence is depicted for KO Sham (F top left), KO injured (F bottom left), WT Sham (F top right), and WT injured (F bottom right) in the right and left hemispheres (blue = DAPI, green = Iba-1). Scale bars = 100μm, 20x. HI = hypoxic-ischemic brain injury.

Area occupied by Iba-1-positive staining was elevated in KO injured animals when compared to uninjured controls, while stain area was not elevated in WT injured animals 

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized U937 (Human histiocytic lymphoma cell line) cells labeling Iba1 with ab178847 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on U937 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab178847 at 1/100 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Iba1 was immunoprecipitated from 1mg of Mouse spleen whole cell lysate with ab178847 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab178847 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Mouse spleen whole cell lysate 10µg (Input).

Lane 2: ab178847 IP in Mouse spleen whole cell lysate.

Lane 3: Rabbit IgG,monoclonal-Isotype Control (ab172730) instead of ab178847 in Mouse spleen whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 5 seconds.

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Iba1 with ab178847 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on microglia of the normal Human cerebrum is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Iba1 antibody ab178847 was used with Tissue Clearing Kit ab243298 to penetrate, stain and clear a 1 mm coronal section of mouse brain. Blue: DAPI , Green: Iba1.

Learn more about tissue clearing kits, reagents, and protocols designed to make it easier to stain thick tissue sections and get more data from each valuable tissue section.

For 1 mm brain sections, we recommend a starting dilution of 1:100, and also using Goat Anti-Rabbit IgG H&L AlexaFluor488 (ab150077) at a dilution of 1:400.

Immunohistochemical analysis of paraffin-embedded Mouse endometrium tissue labeling Iba1 with ab178847 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on macrophages of the mouse endometrium.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized THP-1 (Human monocytic leukemia cell line) cells labeling Iba1 with ab178847 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on THP-1 cell line.

The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab178847 at 1/100 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2: ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.