Recombinant Anti-GFAP antibody [EPR1034Y] (ab68428)

Fluorescence multiplex immunohistochemical analysis of the human cerebellum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (ab254363, green; Opal™520) and anti-P2Y12 (ab254347 red; Opal™570) on human cerebellum. Panel B: anti-P2Y12 stained on microglial cells. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), ab254363 (1/2000 dilution), and ab254347 (1/1000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

Fluorescence multiplex immunohistochemical analysis of the human cerebrum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (ab254363, green; Opal™520) and anti-P2Y12 (ab254347, red; Opal™570) on human cerebrum. Panel B: anti-P2Y12 stained on microglial cells. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), ab254363 (1/2000 dilution), and ab254347 (1/1000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

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.

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.

Immunofluorescence staining of GFAP using ab68428 in primary rat hippocampal mixed glia, (prepared from P2 rat hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. SDPHP4m), DIV4. The cells were fixed with 100% MeOH (5 min), permeabilized with 0.1% Triton-X-100 (in PBS) for 5 mins 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 ab68428 at 0.1 μg/ml and ab4674, Anti-GFAP antibody, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150176, Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown. The antibody ab68428 gave comparable results using 4% formaldehyde fixation (10 min).

Fluorescence multiplex immunohistochemical analysis of human cerebrum tissue (formalin/PFA-fixed paraffin-embedded section). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (ab254363, green; Opal™520) and anti-MAP2 (ab254263, red; Opal™570) on human cerebrum tissue. Panel B: anti-MAP2 stained cell body and dendrites of neurons. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), ab254363 (1/2000 dilution), and ab254263 (1/4000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was used for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

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).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2 µg/mL).<\p>

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

Fluorescence multiplex immunohistochemical analysis of human cerebellum tissue (formalin/PFA-fixed paraffin-embedded section). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (ab254363, green; Opal™520) and anti-MAP2 (ab254263, red; Opal™570) on human cerebellum tissue. Panel B: anti-MAP2 stained cell body and dendrites of neurons. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), ab254363 (1/2000 dilution), and ab254263 (1/4000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was used for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

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.

Fluorescence multiplex immunohistochemical analysis of the Human cerebrum (Formalin/PFA-fixed paraffin-embedded sections).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Opal Polymer HRP Ms + Rb was used as a secondary antibody.DAPI (blue) was used as a nuclear counter stain.

Panel A: Merged staining of anti-GFAP (gray; Opal™690), anti-P2Y12 (green; Opal™520) and anti-MAP2 (red; Opal™570) on human cerebrum.
Panel B: Anti-MAP2 stained cell body and dendrites of neurons.
Panel C: Anti-P2Y12 stained on microglial cells.
Panel D: Anti-GFAP stained on astrocytes.

The section was incubated in three rounds of staining: in the order of ab68428, ab254347, and ab254263 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Fluorescence multiplex immunohistochemical analysis of the Human cerebellum (Formalin/PFA-fixed paraffin-embedded sections).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Opal Polymer HRP Ms + Rb was used as a secondary antibody.DAPI (blue) was used as a nuclear counter stain.

Panel A: Merged staining of anti-GFAP (gray; Opal™690), anti-P2Y12 (green; Opal™520) and anti-MAP2 (red; Opal™570) on human cerebellum.
Panel B: Anti-MAP2 stained cell body and dendrites of neurons.
Panel C: Anti-P2Y12 stained on microglial cells.
Panel D: Anti-GFAP stained on astrocytes.

The section was incubated in three rounds of staining: in the order of ab68428, ab254347, and ab254263 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2 µg/mL).<\p>

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

IHC image of GFAP staining in a section of frozen normal human cerebral cortex performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab68428, 1/250 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. 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 paraffin-embedded mouse cerebral cortex tissue sections labelling GFAP with purified ab68428 at a dilution of 1/500. The secondary antibody used was ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Immunohistochemical analysis of paraffin-embedded human colon tissue sections labelling GFAP with purified ab68428 at a dilution of 1/500. The secondary antibody used was ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. 

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.

Immunohistochemical analysis of paraffin-embedded human cerebral cortex tissue sections labelling GFAP with purified ab68428 at a dilution of 1/500. The secondary antibody used was ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Immunohistochemical analysis of formalin-fixed paraffin-embedded human brain (left) and human glioma (right) tissue sections labelling GFAP with unpurified ab68428 at dilution of 1/250.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse liver tissue sections labelling GFAP with purified ab68428 at a dilution of 1/500. The secondary antibody used was ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0.

Immunohistochemical analysis of formalin-fixed paraffin-embedded mouse brain tissue section labelling GFAP with unpurified ab68428 at dilution of 1/250.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.