Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

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 Sodium citrate (Ph6.0) using retrieval settings of 100°C for 20 minutes. The section was then incubated at room temperature for 1 hour with ab300156 at 1µg/ml dilution (shown in green), ab183830 at 1µg/ml (shown in magenta), and ab302644 at 1µg/ml (shown in yellow). Then incubated for 1 hour with ab150161 Goat F(ab')2 Anti-Rat IgG Fc (Alexa Fluor® 488) preadsorbed 1/1000, ab150083 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed 1/1000, and ab150134 Donkey Anti-Goat IgG H&L (Alexa Fluor® 555) 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 the EVOS™ S1000 Spatial Imaging System (ThermoFisher Scientific) with spectral unmixing and minor subsequent contrast adjustment.

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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling GFAP with ab302644 at 1/1000 (0.998 ug/ml) followed by a Donkey Anti-Goat IgG H&L (HRP) (ab205723) at 1 : 2000 dilution. Positive staining on human cerebrum. The section was incubated with ab302644 for 30 mins at room temperature, the 2nd antibody Donkey Anti-Goat IgG (HRP) ab205723 was used.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is Donkey Anti-Goat IgG H&L (HRP) (ab205723) at 1 : 2000 dilution.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Rat primary neural/glia cell cells labelling GFAP with ab302644 at 1/1000 dilution (0.1ug)/ Right (Red) compared with a Goat IgG / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Donkey anti-Goat IgG (Alexa Fluor® 488, ab150133) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Mouse primary neural/glia cell cells labelling GFAP with ab302644 at 1/1000 dilution (0.1ug)/ Right (Red) compared with a Goat IgG / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Donkey anti-Goat IgG (Alexa Fluor® 488, ab150133) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling GFAP with ab302644 at 1/1000 (0.998 ug/ml) followed by a Donkey Anti-Goat IgG H&L (HRP) (ab205723) at 1 : 2000 dilution. Positive staining on rat cerebrum. The section was incubated with ab302644 for 30 mins at room temperature, the 2nd antibody Donkey Anti-Goat IgG (HRP) ab205723 was used.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is Donkey Anti-Goat IgG H&L (HRP) (ab205723) at 1 : 2000 dilution.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling GFAP with ab302644 at 1/1000 (0.998 ug/ml) followed by a Donkey Anti-Goat IgG H&L (HRP) (ab205723) at 1 : 2000 dilution. Positive staining on mouse cerebrum. The section was incubated with ab302644 for 30 mins at room temperature, the 2nd antibody Donkey Anti-Goat IgG (HRP) ab205723 was used.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is Donkey Anti-Goat IgG H&L (HRP) (ab205723) at 1 : 2000 dilution.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (fresh) tissue labeling GFAP with ab302644 at 1/100 (9.98 ug/ml) dilution followed by ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) at 1/1000 2 ug/mL dilution (Green). Positive staining on mouse cerebrum is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : Secondary antibody is ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488)at 1/1000 2 ug/mL dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum (fresh) tissue labeling GFAP with ab302644 at 1/100 (9.98 ug/ml) dilution followed by ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) at 1/1000 2 ug/mL dilution (Green). Positive staining on rat cerebrum is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : Secondary antibody is ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488)at 1/1000 2 ug/mL dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat primary neural/glia cells labeling GFAP with ab302644 at 1/250 dilution (3.992 μg/ml), followed by ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) antibody at 1/500 dilution (4 μg/mL) (Green). Confocal image showing cytoplasmic staining in rat primary astrocytes. Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. ab10062 Anti-GFAP mouse monoclonal antibody was used to counterstain tubulin at 1/50 dilution (10 μg/ml), followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (Red). The Nuclear counterstain was DAPI (Blue). The negative controls are as follows : -ve control 1 : ab302644 was used as primary antibody at 1/250 dilution, followed by ab150120 at 1 : 1000 dilution (2 μg/ml) 1/1000 dilution (2 μg/mL).  -ve control 2 : ab10062 at 1 : 50 dilution (10 μg/ml) was used as a primary antibody, followed by ab150129 at 1 : 500 dilution (4 μg/ml).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural/glia cells labeling GFAP with ab302644 at 1/250 dilution (3.992 μg/ml), followed by ab150129 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) antibody at 1/500 dilution (4 μg/mL) (Green). Confocal image showing cytoplasmic staining in mouse primary astrocytes. Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. ab10062 Anti-GFAP mouse monoclonal antibody was used to counterstain tubulin at 1/50 dilution (10 μg/ml), followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (Red). The Nuclear counterstain was DAPI (Blue). The negative controls are as follows : -ve control 1 : ab302644 was used as primary antibody at 1/250 dilution, followed by ab150120 at 1 : 1000 dilution (2 μg/ml) 1/1000 dilution (2 μg/mL).  -ve control 2 : ab10062 at 1 : 50 dilution (10 μg/ml) was used as a primary antibody, followed by ab150129 at 1 : 500 dilution (4 μg/ml).

• WB

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Western blot - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (AB302644)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : Lane 1 : 3.25 seconds Lane 2-3 : 70 seconds

All lanes:

Western blot - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Goat IgG (Chimeric) (ab302644) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Mouse cerebellum tissue lysate at 20 µg

Lane 3:

Rat cerebellum tissue lysate at 20 µg

Secondary

All lanes:

Rabbit anti-Goat IgG (H+L), Peroxidase conjugated at 1/5000 dilution

Observed band size: 50 kDa

false

Exposure time: 70s