Multiplex immunohistochemistry - Anti-GFAP antibody [EPR1034Y] (ab68428)

Fluorescence multiplex immunohistochemical analysis of human cerebellum tissue (formalin-fixed paraffin-embedded section).

Merged staining of Neu-N (ab177487; yellow; Opal™570), anti-beta III Tubulin (ab52623; red; Opal™690) and anti-GFAP (ab68428; green; Opal™520).

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ kit.

The section was incubated in three rounds of staining with ab177487 (1/1000 dilution), ab52623 (1/200 dilution) and ab68428 (1/250 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (pH 6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

DAPI (blue) was used as a nuclear counter stain.

Immunoprecipitation - Anti-GFAP antibody [EPR1034Y] (ab68428)

ab68428 at 1/20 dilution immunoprecipitating GFAP in rat brain whole cell lysate observed at 50 KDa (lanes 1 and 2).

Lane 1 (input): Rat brain whole cell lysate 10ug

Lane 2 (+): ab68428 + Rat brain whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab68428 in Rat brain whole cell lysate

For western blotting, ab68428 was used followed by VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

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

All lanes:

Immunoprecipitation - Anti-GFAP antibody [EPR1034Y] (ab68428)

Predicted band size: 49 kDa

Flow Cytometry (Intracellular) - Anti-GFAP antibody [EPR1034Y] (ab68428)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Mouse primary brain cells cells labelling GFAP with ab68428 at 1/500 dilution (0.1ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody [EPR1034Y] (ab68428)

IHC image of GFAP staining in a formalin fixed, paraffin embedded normal human hippocampus tissue section, performed on a Leica Bond™ 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 20 mins. The section was then incubated with ab68428 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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.

Immunocytochemistry/ Immunofluorescence - Anti-GFAP antibody [EPR1034Y] (ab68428)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural mix culture cells labelling GFAP with ab68428 at 1/250 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. ab10062 anti-GFAP antibody [GF5] at 1/100 dilution followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2μg/ml) was used as a counterstain. The Nuclear counterstain was DAPI (Blue).

Western blot - Anti-GFAP antibody [EPR1034Y] (ab68428)

Blocking and Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-GFAP antibody [EPR1034Y] (ab68428) at 1/10000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Human brain tissue lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 49 kDa

Observed band size: 48-50 kDaent signal.