Anti-GFAP antibody - Astrocyte Marker

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GFAP antibody - Astrocyte Marker (AB4674)

ab4674 staining GFAP in primary hippocampal rat neurons/glia (obtained from Neuromics cat. no. PC35101) DIV14. The cells were fixed with 100% methanol (5 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 ab4674 at 1μg/ml and ab87122 Rabbit Poly to Mouse Fructose-bisphosphate aldolase C (No Modifications). Cells were then incubated with ab150176 Goat polyclonal Secondary Antibody to Chicken IgY - H&L (Alexa Fluor^® 594) pre-adsorbed at 1/1000 dilution (shown in red) and ab150081 Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488) pre-adsorbed at 1/1000 dilution (shown in pseudocolour green). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody - Astrocyte Marker (AB4674)

Chromogenic Immunostaining of a formalin fixed paraffin embedded human cerebellum section with chicken pAb to GFAP, dilution 1/20000, detected in DAB (brown) following the ABC method. Hematoxylin (blue) was used as the counterstain. ab4674 detects the core of processes of astrocytes and Bergman glia within the granular and molecular layers. Mouse select image for larger view.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody - Astrocyte Marker (AB4674)

10x and 63x z-stack images of the CA1 hippocampal region for each treatment are shown, with NeuN staining in green and GFAP staining in red.

Two month-old wild-type mice were placed on either control (n = 10) or inhibitor diet (PLX3397, provided at 290 mg/kg chow; n = 14) for 21 d, causing the elimination of approximately 99% of microglia brain-wide.

Fluorescent immunolabeling of the microglia followed a standard indirect technique (primary antibody followed by fluorescent secondary antibody). Brain tissue (sliced at 40 μm) was stained using the anti-ionized calcium-binding adapter molecule 1 (IBA1, polyclonal, rabbit) antibody (1 : 1000; Wako, Cat. #019–19741), mounted on slides, and coverslipped using Dapi Fluoromount-G (SouthernBiotech). Half brain images were obtained by stitching using a Zeiss AxioImager M2 upright microscope and Stereo Investigator software package from MicroBrightField. In addition, tissue was stained with anti-hexaribonucleotide binding protein-3 (NeuN, monoclonal, mouse) antibody (1 : 1000; Millipore; Cat. #MAB377) to label neurons and anti-glial fibrillary acidic protein (GFAP, polyclonal, chicken) antibody (1 : 500; Abcam; Cat. #ab4674) to label astrocytes, and 10x and 63x z-stack images obtained for each treatment using confocal microscopy.

Elmore MR. et al PLoS One. 2015 Apr 7;10(4):e0122912. doi: 10.1371/journal.pone.0122912. eCollection 2015. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GFAP antibody - Astrocyte Marker (AB4674)

ab4674 staining GFAP in primary mouse neurons/glia DIV14 (prepared from E18 mouse hippocampal brain area obtained from Transnetyx Tissue by BrainBits LLC cat.no. C57EHP) cells. The cells were fixed with 100% methanol (5 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 ab4674 at 1µg/ml and ab24345 Mouse mono Anti-L1CAM [2C2]. Cells were then incubated with ab150176 Goat polyclonal Secondary Antibody to Chicken IgY - H&L (Alexa Fluor^® 594) pre-adsorbed at 1/1000 dilution (shown in green) and ab150117 Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody - Astrocyte Marker (AB4674)

IHC image of GFAP staining in a formalin-fixed paraffin-embedded mouse normal brain tissue section.

The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH 6). The section was incubated with ab4674 at 1/1000 dilution for 15 minutes at room temperature. A goat anti-chicken biotinylated secondary antibody was used to detect the primary and visualized using an HRP conjugated ABC system. The section was counterstained with haematoxylin and mounted with DPX.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-GFAP antibody - Astrocyte Marker (AB4674)

Immunofluorescence staining of SOX9 using ab196450 in primary hippocampal mouse neurons/glia (obtained from Transnetyx Tissue by BrainBits LLC cat.no. C57EHP) DIV14. The cells were fixed with 4% formaldehyde (10 min) permeabilized with 0.1% TritonX-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 ab196450 at 1 μg/ml (shown in green), ab4674 (anti-GFAP) at 1/1000 dilution and ab104224 (anti-NeuN) at 1/1000 dilution. Cells were then incubated with ab150176 Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed (shown in red) and ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 647) (shown in purple) all secondary antibodies at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).
As expected most GFAP positive cells are also SOX9 positive while NeuN positive cells are SOX9 negative.
Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GFAP antibody - Astrocyte Marker (AB4674)

Immunofluorescence staining of SOX9 using ab185966 in primary hippocampal mouse neurons/glia (obtained from Transnetyx Tissue by BrainBits LLC cat.no. C57EHP) DIV14. The cells were fixed with 4% formaldehyde (10 min) permeabilized with 0.1% TritonX-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 ab185966 at 1 μg/ml, ab4674 (anti-GFAP) at 1/1000 dilution and ab104224 (anti-NeuN) 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) ab150176 Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed (shown in red) and ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 647) (shown in purple) all secondary antibodies at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).
As expected most GFAP positive cells are also SOX9 positive while NeuN positive cells are SOX9 negative. SOX9 positive cells which are not GFAP positive (e.g. asterisk) are likely neural stem cells/ oligodendrocyte precursor cells present in the culture.
Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-GFAP antibody - Astrocyte Marker (AB4674)

Immunofluorescence staining of SOX9 using ab185230 in primary hippocampal mouse neurons/glia (obtained from Transnetyx Tissue by BrainBits LLC cat.no. C57EHP) DIV14. The cells were fixed with 4% formaldehyde (10 min) permeabilized with 0.1% TritonX-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 ab185230 at 5 μg/ml, ab4674 (anti-GFAP) at 1/1000 dilution and ab104224 (anti-NeuN) 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) ab150176 Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed (shown in red) and ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 647) (shown in purple) all secondary antibodies at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).
As expected most GFAP positive cells are also SOX9 positive while NeuN positive cells are SOX9 negative.
Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

• IHC (PFA fixed)

Supplier Data

Immunohistochemistry (PFA fixed) - Anti-GFAP antibody - Astrocyte Marker (AB4674)

GFAP antibody ab4674 was used with Tissue Clearing Kit ab243298 to penetrate, stain and clear a 500 μm section of rat brain. Learn more about tissue clearing kits, reagents, and protocols designed to make it easier to stain thick tissue sections and get more data from each valuable tissue section.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP antibody - Astrocyte Marker (AB4674)

IHC image of GFAP staining in a formalin fixed paraffin embedded normal rat hippocampus tissue section.

The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH 6). The section was incubated with ab4674 at 1/1000 dilution for 15 minutes at room temperature. A goat anti-chicken biotinylated secondary antibody was used to detect the primary and visualized using an HRP conjugated ABC system. The section was counterstained with haematoxylin and mounted with DPX.

• IHC-FrFl

Supplier Data

Immunohistochemistry - Free Floating - Anti-GFAP antibody - Astrocyte Marker (AB4674)

Immunofluorescent analysis of a section of mouse hippocampus stained with ab4674 at a 1 : 5000 dilution in green.

Costained with a rabbit pAb to FOX3/NeuN dilution 1 : 5000 in red. The blue is DAPI staining of nuclear DNA. Following transcardial perfusion with 4% paraformaldehyde mouse brain was post fixed for 24 hours cut to 45 μM and free-floating sections were stained. The GFAP antibody stains a network of astroglial cells while the Fox3/NeuN antibody stains the nuclei and proximal perikarya of neurons.

• WB

Supplier Data

Western blot - Anti-GFAP antibody - Astrocyte Marker (AB4674)

All lanes:

Western blot - Anti-GFAP antibody - Astrocyte Marker (ab4674) at 1/5000 dilution

Lane 1:

Rat whole brain lysate

Lane 2:

Mouse whole brain lysate

Predicted band size: 49 kDa

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