Anti-Iba1 antibody [EPR16589] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

This data was developed using ab178847, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain tissue* labelling Iba1 with ab178847 at 1ug/ml followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining in human Alzheimer's brain.

The section was incubated with ab178847 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0 epitope retrieval solution1) for 20 mins.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178847).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178847).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178847).

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

This data was developed using ab178847, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (perfused fixed) tissue labeling Iba1 with ab178847 at 1200 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (ab150081) at 1/1000 dilution (Green).

Confocal image showing positive staining on mouse liver. The nuclear counterstain was DAPI (Blue). The section was incubated with ab178847 for 60 mins at room temperature. The section was then mounted using Fluoromount^®.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (ab150081) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC (PFA fixed)

Supplier Data

Immunohistochemistry (PFA fixed) - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

This data was developed using ab178847, the same antibody clone in a different buffer formulation. 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178847).

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

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

This data was developed using ab178847, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum (perfused fixed) tissue labeling Iba1 with ab178847 at 1200 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (ab150081) at 1/1000 dilution (Green).

Confocal image showing positive staining on rat cerebrum. The nuclear counterstain was DAPI (Blue). The section was incubated with ab178847 for 60 mins at room temperature. The section was then mounted using Fluoromount^®.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (ab150081) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

This data was developed using ab178847, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (perfused fixed) tissue labeling Iba1 with ab178847 at 1200 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (ab150081) at 1/1000 dilution (Green).

Confocal image showing positive staining on mouse cerebrum. The nuclear counterstain was DAPI (Blue). The section was incubated with ab178847 for 60 mins at room temperature. The section was then mounted using Fluoromount^®.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (ab150081)at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

This data was developed using ab178847, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat liver (perfused fixed) tissue labeling Iba1 with ab178847 at 1200 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (ab150081) at 1/1000 dilution (Green).

Confocal image showing positive staining on rat liver. The nuclear counterstain was DAPI (Blue). The section was incubated with ab178847 for 60 mins at room temperature. The section was then mounted using Fluoromount^®.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (ab150081) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

Immunohistochemical analysis of paraffin-embedded Rat 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 rat cerebrum is observed.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178847).

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

• IP

Supplier Data

Immunoprecipitation - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178847).

All lanes:

Immunoprecipitation - Anti-Iba1 antibody [EPR16589] - Microglia marker (<a href='/en-us/products/primary-antibodies/iba1-antibody-epr16589-microglia-marker-ab178847'>ab178847</a>)

Predicted band size: 16 kDa

false

• WB

Lab

Western blot - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

This data was developed using ab178847, the same antibody clone in a different buffer formulation. Different batches of ab178847 were tested on U-937 (Human histiocytic lymphoma monocyte) lysate at 0.5 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 17 kDa.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (<a href='/en-us/products/primary-antibodies/iba1-antibody-epr16589-microglia-marker-ab178847'>ab178847</a>)

Predicted band size: 16 kDa

false

• WB

Supplier Data

Western blot - Anti-Iba1 antibody [EPR16589] - BSA and Azide free (AB221790)

This data was developed using ab178847, the same antibody clone in a different buffer formulation. 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.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (<a href='/en-us/products/primary-antibodies/iba1-antibody-epr16589-microglia-marker-ab178847'>ab178847</a>) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Mouse hippocampus tissue at 20 µg

Lane 4:

Rat spleen tissue lysate at 20 µg

Lane 5:

Rat brain tissue lysate at 20 µg

Lane 6:

Rat hippocampus tissue lysate at 20 µg

Secondary

All lanes:

Western blot - PE Anti-ENPP3/B10 antibody [NP4D6] (<a href='/en-us/products/primary-antibodies/pe-enpp3-b10-antibody-np4d6-ab90751'>ab90751</a>) at 1/20000 dilution

Predicted band size: 16 kDa

Observed band size: 17 kDa

false

Exposure time: 40s