Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse cerebrum tissue labeling GFAP with ab278054 at 1/700 (0.938 µg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at a 1/1000 dilution. Nuclear counterstain is DAPI. Positive staining on mouse cerebrum. 

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) used at a 1/1000 dilution.

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

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling GFAP with ab278054 at 1/2000 (0.235 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human cerebrum. The section was incubated with ab278054 for 30 mins at room temperature. 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural/glia cells labelling GFAP with ab278054 at 1/500 (0.938 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (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 at 1/200 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) at a 1/500 dilution (Red) to counterstain. The nuclear counterstain was DAPI (Blue).

Negative control 1: ab278054 at a 1/500 dilution followed by ab150120 at a 1/500 dilution.

Negative control 2: ab10062 at a 1/500 dilution followed by ab150077 at a 1/1000 dilution.

Flow Cytometry analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized mouse primary brain cells labeling GFAP using ab278054 at a 1/500 dilution (0.1 µg) (Right panel) compared to Rabbit monoclonal IgG (ab172730) (Left panel).

Secondary antibody is Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) used at a 1/2000 dilution.

All lanes : Anti-GFAP antibody [RM1003] (ab278054) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 49 kDa
Observed band size: 40-54 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

The molecular weight observed is consistent with the literature (PMID: 25975510).

Blocking/Dilution buffer: 5% NFDM/TBST.

GFAP was immunoprecipitated from 0.35 mg mouse brain lysate with ab278054 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab278054 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain lysate 10 μg.
Lane 2: ab278054 IP in mouse brain lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab278054 in mouse brain lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 seconds.
The molecular weight observed is consistent with the literature (PMID: 824020; PMID:2294; PMID: 6340792).

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling GFAP with ab278054 at 1/2000 (0.235 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse cerebrum. The section was incubated with ab278054 for 30 mins at room temperature. 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

All lanes : Anti-GFAP antibody [RM1003] (ab278054) at 1/1000 dilution

Lane 1 : Human brain lysate
Lane 2 : Human liver lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 49 kDa
Observed band size: 40-54 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Negative control: Human liver. (PMID: 25975510).
The molecular weight observed is consistent with the literature (PMID: 25975510).

Blocking/Diluting buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling GFAP with ab278054 at 1/2000 (0.235 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat cerebrum. The section was incubated with ab278054 for 30 mins at room temperature. 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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