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.

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.

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.

Immunofluorescent analysis of a section of mouse hippocampus stained with ab4674 at a 1:5,000 dilution in green.

Costained with a rabbit pAb to FOX3/NeuN dilution 1:5,000, 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.

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.

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.