Anti-Iba1 抗体 [EPR16589]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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

Stephanie R. Beldick et al.,PLOS ONE.,Fig 5.; doi.org/10.1371/journal.pone.0208105.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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.

• IHC (PFA fixed)

Supplier Data

Immunohistochemistry (PFA fixed) - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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] - Microglia marker (AB178847)

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] - Microglia marker (AB178847)

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).

• IP

Supplier Data

Immunoprecipitation - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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.

All lanes:

Immunoprecipitation - Anti-Iba1 antibody [EPR16589] - Microglia marker (ab178847)

Predicted band size: 16 kDa

false

• WB

Supplier Data

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (ab178847) at 1/1000 dilution

Lane 1:

MOLT-4 (Human lymphoblastic leukemia cell line) whole cell lysate at 10 µg

Lane 2:

U937 (Human histiocytic lymphoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 16 kDa

Observed band size: 17 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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 (ab178847)

Predicted band size: 16 kDa

false

• WB

Supplier Data

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (ab178847) at 1/2000 dilution

Lane 1:

Human spleen lysate at 20 µg

Lane 2:

THP-1 (Human monocytic leukemia cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 16 kDa

Observed band size: 17 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

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 (ab178847) 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='/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

• WB

Supplier Data

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (AB178847)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 and 2 : 1 minute; Lane 3 and 4 : 3 minutes.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16589] - Microglia marker (ab178847) at 1/1000 dilution

Lane 1:

Mouse spleen lysate at 10 µg

Lane 2:

Rat spleen lysate at 10 µg

Lane 3:

Mouse testis lysate at 10 µg

Lane 4:

Rat testis lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 16 kDa

Observed band size: 17 kDa

false