Anti-Iba1 antibody [EPR16588] - Microglia marker

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

IHC image of Iba1 staining in human normal hippocampus formalin fixed paraffin embedded tissue section performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6 epitope retrieval solution 1) for 20 mins. The section was then incubated with ab178846 at 1/2000 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions primary antibody concentration and antibody incubation times.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Chromogenic multiplex immunohistochemical staining of FFPE normal human cerebellum tissue. ab177487, anti-NeuN DAB chromogen. ab68428, anti-GFAP purple chromogen and ab178846, anti- Iba1 teal chromogen plus haematoxylin counterstain. Chromogenic immunostaining was performed on a Roche Ventana Discovery Ultra instrument. The section was deparaffinised and incubated with CC1 solution for 24min 100°C. Following this with 3 rounds of staining in the order of ab177487 (1/600), ab178846 (1/4000) ab68428 (1/1000). Between rounds of staining, antibody denaturation was conducted using Ultra CC2 solution for 8min at 100°C to avoid cross reactivity. Signal was developed with anti-rabbit HQ followed by anti-HQ HRP coupled with Chromomap DAB kit, Discovery purple or Discovery teal chromogens and haematoxylin II counterstain.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Immunohistochemical analysis of formalin fixed paraffin embedded human cerebellum labelling Iba1 with ab178846 at a dilution of 1/4000. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an ChromoMap DAB (RUO) IHC Detection Kit with anti rabbit HQ and anti HQ HRP. Heat mediated antigen retrieval was conducted for 24 min with DISCOVERY cell conditioning solution (CC1)  100°C, pH 8.5. ab178846 was incubated at 37°C for 16 min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Composite multiplex immunofluorescence staining of Iba1, GFAP and MAP2 staining in a section of formalin-fixed paraffin-embedded human cerebral cortex*.

Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with EDTA (Ph9.0) using retrieval settings of 100°C for 40 minutes. The section was then incubated at room temperature for 1 hour with ab323239 at 5µg/ml dilution (shown in green), ab300645 at 5µg/ml (shown in magenta), and ab178846 at 5µg/ml (shown in yellow). Then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed 1/1000, ab150084 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed 1/1000, and ab150173 Goat Anti-Chicken IgY H&L (Alexa Fluor® 488) preadsorbed 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Dako Fluorescence Mounting Medium ^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

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

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum (perfused fixed) tissue labeling Iba1 with ab178846 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 ab178846 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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Flow cytometry overlay histogram showing left Raw264.7 positive cells and right negative NIH3T3 stained with ab178846 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab178846) (1x 106 in 100μl at 0.2μg/ml (1/9850 dilution)) for 30min at 22°C.The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°CIsotype control antibody (black line) was Recombinant Rabbit IgG monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

AbReview63039****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Formaldehyde-fixed paraffin-embedded cynomolgus monkey brain tissue stained for Iba1 using ab178846 at 1/6000 dilution in immunohistochemical analysis.

Antigen Retrieval : Heat mediated - Buffer/Enzyme Used : pH 9.0 EDTA

This image was courtesy of an annonymous Abreview

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (perfused fixed) tissue labeling Iba1 with ab178846 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 ab178846 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-tissue clearing

Supplier Data

IHC-tissue clearing - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Anti-Iba1 ab178846 was used with Tissue clearing kit – CUBIC (ab316246) and 3D Tissue Staining Kit – CUBIC (ab316248) to penetrate stain and clear a whole mouse brain.

Learn more about tissue clearing kits reagents and protocols designed to make it easier to stain whole brains and get more data from each valuable tissue sample.

For a whole mouse brain we recommend starting with 3.5 ug of ab178846 and using a Fab fragment secondary antibody with 2.34 µg to create an antibody complex before 3D staining (see protocol for details). Additive A was used during the staining process.

The sample was imaged using a light-sheet microscope.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (perfused fixed) tissue labeling Iba1 with ab178846 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 ab178846 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).

• WB

Supplier Data

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration :

5% NFDM /TBST.

Based on sequence analysis, ab178846 recognizes 2 isoforms with the predicted MWs of 17KDa and 11KDa, respectively.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (ab178846) at 1/2000 dilution

All lanes:

HL-60 (human promyelocytic leukemia cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 16 kDa

Observed band size: 10 kDa,15 kDa

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• WB

Lab

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Abcam recommends blocking in milk for cleaner blots with reduced background, in comparison to BSA.

This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab178846 (anti-Iba1 antibody; 1/500 dilution) for 18 hours at 4°C. Antibody binding was detected using ab97040 (HRP-labelled goat anti-mouse IgG) at 1 : 50,000 dilution for 1 hour at room temperature and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (ab178846) at 1/500 dilution

Lane 1:

Human Iba1 recombinant protein at 0.1 µg

Lane 2:

HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 3:

A431 (human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embyro fibroblast cell line) whole cell lysate at 30 µg

Lane 5:

Human spleen tissue lysate at 20 µg

Lane 6:

Mouse spleen tissue lysate at 30 µg

Lane 7:

Rat spleen tissue lysate at 30 µg

Lane 8:

U937 (human histiocytic lymphoma cell line) whole cell lysate at 30 µg

Lane 9:

MOLT-4 (human lymphoblastic leukemia cell line) whole cell lysate at 20 µg

Lane 10:

THP-1 (human monocytic leukemia cell line) whole cell lysate at 30 µg

Lane 11:

THP-1 whole cell lysate, PMA treated at 30 µg

Lane 12:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 30 µg

Lane 13:

C6 (rat glial tumor cell line) whole cell lysate at 30 µg

Lane 14:

NR8383 whole cell lysate at 30 µg

Predicted band size: 16 kDa

true

Exposure time: 1min

• WB

Lab

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Blocking buffer and concentration : 5% NFDM/TBST. Diluting buffer and concentration : 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. ab181602 was used as loading control.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (ab178846) 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 lysate 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 - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 16 kDa

Observed band size: 17 kDa

false

Exposure time: 40s

• WB

Unknown

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

All lanes:

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (ab178846)

Lane 1:

Mouse testis

Lane 2:

Mouse liver

Lane 3:

Rat testis

Lane 4:

Rat liver

Predicted band size: 16 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

IHC image of Iba1 staining in rat normal brain formalin fixed paraffin embedded tissue section performed on a Leica Bond™ system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6 epitope retrieval solution 1) for 20 mins. The section was then incubated with ab178846 at 1/2000 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions primary antibody concentration and antibody incubation times.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Immunofluorescence staining of Iba-1 using ab178846 in primary rat hippocampal mixed glia (prepared from P2 rat hippocampal brain area obtained from Transnetyx Tissue by BrainBits LLC cat.no. SDPHP4m) DIV4. The cells were fixed with 4% formaldehyde (10 min) permeabilized with 0.1% Triton-X-100 (in PBS) for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab178846 at 0.1 μg/ml and ab4674 Anti-GFAP antibody at 1/1000 dilution. Cells were then incubated with ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150176 Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown. The antibody ab178846 gave comparable results using MeOH fixation (100% 5 min).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling Iba1 with ab178846 at a 1/2000 dilution showing cytoplasm and nuclear staining on Glial cells. Counter stained with hematoxylin. Prediluted HRP Polymer for Rabbit/Mouse IgG was used as the secondary aantibody. Negative control also shown.

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

0.1% Triton-X 100 permeabilized paraformaldehyde-fixed Mouse cell Microglia cells labeling Iba1 (green) using ab178846 at 1/500 dilution in ICC/IF analysis.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

IHC image of Iba1 staining in mouse normal brain formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab178846, 1/2000 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Intracellular Flow Cytometry analysis of 2% paraformaldehyde fixed U937 (human histiocytic lymphoma cell line) cells labeling Iba1with ab178846 at 1/160 dilution (red line). Secondary antibody used is a goat anti rabbit IgG (FITC) at 1/150 dilution. The isotype control is rabbit monoclonal IgG (black line). The unlabeled control is cells without incubation with primary and secondary antibodies (blue line).

• WB

Lab

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Different batches of ab178846 were tested on THP-1 (Human monocytic leukemia monocyte) lysate at 0.02 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 15 kDa.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (ab178846)

Predicted band size: 16 kDa

false

• WB

Supplier Data

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (AB178846)

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM /TBST

All lanes:

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (ab178846) at 1/10000 dilution

Lane 1:

THP-1 (human monocytic leukemia cell line) whole cell lysate at 10 µg

Lane 2:

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

Lane 3:

Human spleen whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 16 kDa

Observed band size: 15 kDa

true