Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Mouse IgG1 (Chimeric)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Mouse IgG1 (Chimeric) (AB279289)

IHC image of GFAP staining in a section of formalin-fixed paraffin-embedded normal human cerebral cortex 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 (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab279289, 1μg/ml, for 15 mins at room temperature. A rabbit anti-mouse IgG1, ab125913, was added for 8 mins at room temperature and detected using an HRP conjugated goat anti-rabbit compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

The inset secondary-only control 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Mouse IgG1 (Chimeric) (AB279289)

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized rat primary neural/glia cells labelling GFAP with ab279289 at 1/1000 dilution (0.1μg)/ Right compared with a Mouse monoclonal IgG isotype control/ Left.

Goat Anti-Mouse IgG (Alexa Fluor^® 647, ab150119) at 1/2000 dilution was used as the secondary antibody.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Mouse IgG1 (Chimeric) (AB279289)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum tissue labeling GFAP with ab279289 at /500 (1.968 μg/ml) dilution followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on mouse cerebrum is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor^{® 4}88)at 1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• ICC

Lab

Immunocytochemistry - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Mouse IgG1 (Chimeric) (AB279289)

ab279289 staining GFAP in primary hippocampal rat neurons/glia (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes 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 ab279289 at 1μg/ml and ab6046, Rabbit polyclonal to beta Tubulin. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150084, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
The antibody ab279289 gave comparable results using MeOH fixation (100%, 5 min).

• IP

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Immunoprecipitation - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Mouse IgG1 (Chimeric) (AB279289)

GFAP was immunoprecipitated from 0.35 mg rat brain tissue lysate 10 μg with ab279289 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab279289 at 1/1000 dilution. mouse IgG for IP (HRP) (ab131368) was used at 1/5000 dilution.

Lane 1 : Rat brain tissue lysate 10μg.

Lane 2 : ab279289 IP in rat brain tissue lysate.

Lane 3 : Mouse monoclonal IgG1 (ab18443) instead of ab279289 in rat brain tissue lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 23 seconds.

All lanes:

Immunoprecipitation - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Mouse IgG1 (Chimeric) (ab279289)

Predicted band size: 49 kDa

false

• WB

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Western blot - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Mouse IgG1 (Chimeric) (AB279289)

Blocking/Dilution buffer : 5% NFDM/TBST.

The molecular weight observed is consistent with the literature (PMID : 824020, 2294, 6340792).

Exposure times : Lane 1 : 3.25 seconds; Lane 2 : 48 seconds; Lane 3 : 10 seconds.

All lanes:

Western blot - Anti-GFAP antibody [EPR1034Y] - Astrocyte marker - Mouse IgG1 (Chimeric) (ab279289) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 49 kDa

Observed band size: 40-50 kDa

false