Recombinant Anti-GFAP antibody [EPR1034Y] - 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.

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.

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).

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).

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.

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.