Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 cells labelling Iba1 with ab289874 at 1/100 dilution, followed by Donkey Anti-Goat IgG H&L (Alexa Fluor^®488) (ab150129) antibody at 1/500 dilution (Green).

Confocal image showing cytoplasmic and membranous staining in THP-1 cell line. Negative control : MCF7 (PMID : 30386176).

was used to counterstain tubulin at 1/200 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is Donkey Anti-Goat IgG H&L (Alexa Fluor^®488) (ab150129) at 1/500 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

IHC image of ab289874 staining Iba1 in FFPE Human Skeletal Muscle tissue (Negative Tissue).*

Performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20mins. The section was then incubated with ab289874 at 1µg/ml, for 15 mins at room temperature and detected using ab205723 Donkey Anti-Goat IgG H&L (HRP). DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only image is taken from an identical assay without primary antibody.

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.

*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] - Microglia marker - Goat IgG (Chimeric) (AB289874)

IHC image of ab289874 staining Iba1 in FFPE Human Glioblastoma tissue.*

Performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20mins. The section was then incubated with ab289874 at 1µg/ml, for 15 mins at room temperature and detected using ab205723 Donkey Anti-Goat IgG H&L (HRP). DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only image is taken from an identical assay without primary antibody.

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.

*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] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Iba1 with ab289874 at 1/1000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on microglia in the human cerebrum. The section was incubated with ab289874 for 30 mins at room temperature and followed by specific anti-goat IgG antibody (ab97101) for 8 mins. 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Iba1 with ab289874 at 1/1000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on Kupffer cells in the human liver. The section was incubated with ab289874 for 30 mins at room temperature and followed by specific anti-goat IgG antibody (ab97101) for 8 mins. 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt

Lab

Flow Cytometry - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Flow cytometric analysis of 4% Paraformaldehyde fixed, 90% Methanol permeabilized MCF7 (Human breast adenocarcinoma epithelial cell) / THP-1 (Human monocytic leukemia monocyte) cells labelling Iba1 with ab289874 at 1/1000 compared with a Goat IgG /Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Donkey anti-Goat IgG (Alexa Fluor^®488, ab150133) at 1/2000 was used as the secondary antibody.

Negative control :  MCF7 (PMID : 30386176).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Iba1 with ab289874 at 1/1000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on human tonsil. The section was incubated with ab289874 for 30 mins at room temperature and followed by specific anti-goat IgG antibody (ab97101) for 8 mins. 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Iba1 with ab289874 at 1/1000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on Kupffer cells in the mouse liver.

The section was incubated with ab289874 for 30 mins at room temperature and followed by specific anti-goat IgG antibody (ab97101) for 8 mins. 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural/glia cell cells labelling Iba1 with ab289874 at 1/1000 dilution , followed by ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor^® 488) antibody at 1/500 dilution (Green).

Confocal image showing positive staining in mouse primary microglial cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).
Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

Rabbit anti-Iba1 antibody was used to counterstain Iba1 at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor^® 488) at 1/500 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling Iba1 with ab289874 at 1/1000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on microglia in the mouse cerebrum. The section was incubated with ab289874 for 30 mins at room temperature and followed by specific anti-goat IgG antibody (ab97101) for 8 mins. 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat primary neural/glia cell cells labelling Iba1 with ab289874 at 1/1000 dilution, followed by ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor^® 488) antibody at 1/500 dilution (Green).

Confocal image showing positive staining in rat primary microglial cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).
Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

Rabbit anti-Iba1 antibody was used to counterstain Iba1 at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor^® 488) at 1/500 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling Iba1 with ab289874 at 1/1000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on microglia in the rat cerebrum. The section was incubated with ab289874 for 30 mins at room temperature and followed by specific anti-goat IgG antibody (ab97101) for 8 mins. 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Iba1 with ab289874 at 1/1000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on Kupffer cells in the rat liver. The section was incubated with ab289874 for 30 mins at room temperature and followed by specific anti-goat IgG antibody (ab97101) for 8 mins. 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• WB

Lab

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (AB289874)

Western blot : Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (ab289874) at 1/1000, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 17 kDa. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Donkey anti-Goat IgG 800CW and Donkey anti-Mouse IgG 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Iba1 antibody [EPR16588] - Microglia marker - Goat IgG (Chimeric) (ab289874) at 1/1000 dilution

Lane 1:

THP-1 (Human monocytic cell line) cell lysate

Lane 2:

Human spleen tissue lysate

Lane 3:

Human brain tissue lysate

Lane 4:

MCF7 (human breast cancer cell line) cell lysate

Lane 5:

RAW264.7 (mouse macrophage cell line) cell lysate

Lane 6:

Mouse spleen tissue lysate

Lane 7:

Mouse brain tissue lysate

Lane 8:

NIH/3T3 (mouse embryonic fibroblast cell line) cell lysate

Lane 9:

Rat spleen tissue lysate

Lane 10:

Rat brain tissue lysate

Lane 11:

NR8383 (rat alveolar macrophage cell line) cell lysate

Lane 12:

C6 (rat glioma cell line) cell lysate

Secondary

All lanes:

Donkey anti-Goat IgG 800CW and Donkey anti-Mouse IgG 680RD at 1/20000 dilution

Observed band size: 17 kDa,36 kDa

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