Anti-Iba1 antibody [EPR16588] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178846).

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

This data was developed using ab178846, 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 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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

This data was developed using ab178846, 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 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).

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

This data was developed using ab178846, 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 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).

• WB

Lab

Western blot - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

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. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178846).

All lanes:

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker (<a href='/en-us/products/primary-antibodies/iba1-antibody-epr16588-microglia-marker-ab178846'>ab178846</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 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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178846).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178846).

Heat mediated antigen retrieval was performed 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 [EPR16588] - BSA and Azide free (AB220815)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178846).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178846).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

Clone EPR16588 (ab220815) has been successfully conjugated by Abcam. This image was generated using Anti-Iba1 antibody [EPR16588] (Alexa Fluor® 488). Please refer to ab225260 for protocol details.

IHC image of Iba1 staining in a section of formalin-fixed paraffin-embedded normal human tonsil*.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Biocare Medical NxGen pressure cooker using retrieval settings of 110^oC for 20 minutes. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature.  The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab225260 at 1/100 dilution (shown in green) and counterstained using ab195884, Rat monoclonal to Tubulin (Alexa Fluor^® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

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-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178846).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178846).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

Clone EPR16588 (ab220815) has been successfully conjugated by Abcam. This image was generated using Anti-Iba1 antibody [EPR16588] (Alexa Fluor® 647). Please refer to ab225261 for protocol details.

IHC image of Iba1 staining in a section of formalin-fixed paraffin-embedded normal human tonsil*.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Biocare Medical NxGen pressure cooker using retrieval settings of 110^oC for 20 minutes. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature.  The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab225261 at 1/100 dilution (shown in red) and counterstained using ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

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.

• WB

Lab

Western blot - Anti-Iba1 antibody [EPR16588] - BSA and Azide free (AB220815)

This data was developed using ab178846, the same antibody clone in a different buffer formulation. 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 (<a href='/en-us/products/primary-antibodies/iba1-antibody-epr16588-microglia-marker-ab178846'>ab178846</a>)

Predicted band size: 16 kDa

false