Recombinant Anti-GFAP antibody [EPR1034Y] - Rat IgG2a (Chimeric) (ab279291)

Immunocytochemical analysis of 4% PFA-fixed, 0.1% Triton X-100 permeabilized mouse primary neural/glia tissue labeling GFAP with ab279291 at 1/50 (17 µg/ml) dilution followed by ab150161 Goat Anti-Rat IgG (Alexa Fluor® 488) pre-adsorbed at 1/1000 dilution (Green). Positive staining on mouse primary astrocytes is observed. The nuclear counterstain was DAPI (Blue).

Counterstain: ab207165 anti-GFAP rabbit mAb at 1/1000 dilution with secondary antibody ab150088 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) pre-adsorbed at 1/1000 dilution.

Negative controls: ab279291 at 1/50 dilution with secondary ab150080 at 1/500 dilution; and ab207165 at 1/1000 dilution with secondary ab150161 at 1/1000 dilution

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1: 3.25 seconds; Lane 2, 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 ab279291, 1µg/ml, for 15 mins at room temperature. A rabbit anti-rat IgG2a, ab102248, 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 ab279291 at 1/500 (2.01 µg/ml) dilution followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution. The nuclear counterstain was DAPI (Blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Positive staining on mouse cerebrum is observed.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized rat primary neural/glia cells labelling GFAP with ab279291 at 1/1000 dilution (0.1µg)/ Right compared with a Rat monoclonal IgG isotype control/ Left.

Goat F(ab)2 Anti-Rat IgG Fc (Alexa Fluor® 647, ab150163) at 1/2000 dilution was used as the secondary antibody.