Glial fibrillary acidic protein (gfap)

Immunohistochemistry staining for brain was performed on fixed frozen section as previously described43 (link), with primary antibodies: anti-P2X7 receptor (Millipore, Temecula, CA), anti-GFAP (Abcam, Cambridge, MA), anti–ZO-1 (Invitrogen, Grand Island, NY), and anti-vWF (Santa Cruz Biotechnology, Santa Cruz, CA), and followed by fluorescein isothiocyanate-conjugated and Texas Red-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA). The colocalization of P2X7 receptor with GFAP or vWF and the colocalization of ZO-1 with vWF were examined by a confocal microscope (LSM780, Zeiss, Jena, Germany).

For IF (N = 3-4 per genotype and diet, 1-2 per sex), one brain hemisphere was fixed in 4% PFA and sucrose before freezing in cold 2-methylbutene and slicing at 30 μm. IF stains were performed for Iba1 (Wako, Cat #: 019-19741), GFAP (Cell Signaling, Cat #: 3670S), Doublecortin (DCX, ThermoFisher, Cat #: 481200), NeuN (Chemicon, Cat #: MAB377), and cFOS (Abcam, Cat # ab190289). Stains were imaged using a Zeiss AxioSkop at either 10× (Iba1, GFAP, and cFOS) or 20× (co-stains and DCX) magnification. Staining was analyzed using Image J, with quantification from 4 brain slices/mouse. Hippocampal images were taken from the stratum radiatum and stratum oriens of CA1 and CA3, and the CA4 and the molecular layers (MO) of the dentate gyrus (DG). They were also taken from the arcuate nucleus, the dorsal medial, and lateral hypothalamus, and from multiple layers of the cerebral cortex (Fig. 1). Iba1 and GFAP were analyzed as the percent area covered by IF staining in the image. DCX and cFOS expression were analyzed as the counts of antibody positive cells. All quantification was conducted blinded.
Fig. 1

Illustration of mouse brain hemisphere with areas analyzed for IF outlined

Rats were deeply anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde prepared in phosphate buffer saline (PBS) buffer. Whole brain was collected and incubated in 4% paraformaldehyde for 16 h, transferred to 30% sucrose prepared in PBS, and stored at 4 °C for at least 48 h before tissue processing. Each brain was sectioned in 40-µm-thick coronal slices. Sections containing striatum were stained with anti-Iba1 (Wako: 019-19741; diluted 1:500), anti-glial fibrillary acidic protein (GFAP; Cell Signaling Technology: #3670; diluted 1:300), or anti-pCREB (Millipore: 06-519; diluted 1:500) antibodies.
Sections were incubated with secondary antibody goat anti-rabbit conjugated to Alexa Fluor 594 (Iba1) and goat anti-mouse conjugated to Alexa 488 (GFAP and pCREB), both diluted 1:200 in blocking buffer (0.3% Goat serum, 0.3% Triton X-100, 0.2% Tween, 10% 10X PBS in water). Sections were imaged on a Zeiss fluorescence confocal microscope with a 25 × objective and 0.5 × Zoom for GFAP and pCREB images and 10 × objective and 0.6 × Zoom for Iba1 images and analyzed with ImageJ software. Quantitation of fluorescence was performed in the dorsomedial area around the striatal lesion and analyzed as percent area of antibody staining (GFAP and IBA1) or intensity (cumulative distribution and average intensity; pCREB).