Chicken anti gfap

Primary mixed cortical cultures of glial cells from 3-day-old pups were generated and microglia were fluorescently labeled and sorted as previously described [18 (link)]. In brief, 17 days after plating, cultures were dissociated (HyClone Trypsin .25%; Thermo Scientific) and resuspended in FACS buffer: HBSS (Gibco; Invitrogen 14025) supplemented with 2% BSA (Sigma-Aldrich, A7906) and containing 1 U/μl SUPERase In™ RNase Inhibitor (Ambion; Life Technologies, AM2694). Cells were centrifuged at 1305 g for 5 min and suspended in 50 μl of fresh FACS buffer to wash. The cells were stained for 1 h at 4 °C with chicken anti-GFAP (Abcam, ab4674) to label astrocytes and rabbit anti-IBA1 (Wako, 016-20001) to label microglial cells. Cells were washed three times and incubated for 30 min at 4 °C with secondary antibodies: donkey anti-rabbit 647 (Invitrogen, A31573) and goat anti-chicken 488 (Invitrogen, A11039). Samples were re-suspended in 200 μl of FACS buffer and sorted on BD Biosciences LSR II SORP. Purified microglia were collected separately and stored in RLT Buffer (QIAGEN, 79216) at − 80 °C. Total RNA was isolated (QIAGEN, 74104) from purified samples from D2.C3ar1^−/− and D2.C3ar1^+/+ mice.

Paraffin-embedded tissue was sectioned with a thickness of 6 μm. One slide per animal was used for staining, each containing 5 equally spaced sections. Cresyl violet staining was performed to visualize neurons^{22 (link)}. Antibody staining was performed as previously described^{114 (link)}, except that all primary antibodies were incubated overnight at RT, followed by another overnight at 4 °C. For Aβ staining, the Mouse On Mouse detection kit (Vector labs) was used according to manufacturer’s instructions. The following primary antibodies were used: mouse anti-human Aβ (1:150; Covance); chicken anti-GFAP (1:150; Abcam). Cy2/Cy3-conjugated anti-mouse/chicken secondary antibodies (1:150; Jackson Immunoresearch) were used. For counterstaining, 4’,6-diamidino-2-phenylindole (1:5000; Biolegend) was used.

Immunohistochemistry was performed as follows. The rats’ DRG were removed and placed in 4 % paraformaldehyde for 2 h at room temperature. The DRG were washed in 0.1 M PBS prior to incubation in 20 % sucrose in PBS overnight. Then, 12-μm-thick sections were cut using a cryostat (Leica) and mounted onto the slides. The sections were washed in PBS and incubated in blocking solution containing 3 % bovine serum albumin (BSA) in PBS with 0.3 % Triton X-100 for 30 min at room temperature. Primary antibodies against glial fibrillary acidic protein (GFAP) (chicken anti-GFAP, Abcam) and P2X₇ (rabbit anti-P2X₇, Abcam) were diluted 1:100 in PBS containing 1 % BSA and incubated overnight at 4 °C. The sections were washed in PBS and incubated with the secondary antibody [goat anti-rabbit TRITC (tetraethyl rhodamine isothiocyanate) (Jackson ImmunoResearch Inc., West Grove, PA, USA) and goat anti-chicken FITC (Beijing Zhongshan Biotech CO.)] diluted 1:200 in PBS for 45 min at 37 °C. Finally, the sections were washed in PBS. Controls omitted the primary antibody. The sections were imaged using a fluorescence microscope (Olympus, Tokyo, Japan). Data were collected from six animals in each group. Five fields randomly selected that contained approximately 50 neurons each were analyzed from each animal and averaged.

Tissue sections were rinsed in PBS-0.2% Triton X-100 (PBST) solution. Non-specific binding was blocked with 10% normal donkey serum (NDS) in a PBST solution for 60 min at room temperature, followed by overnight incubation at 4°C with primary antibodies diluted in 1% NDS-PBST (1:2000 for mouse anti-GFAP, Sigma; 1:1000 for chicken anti-GFAP, Abcam; 1:1000 for anti-Iba-1, Wako; 1:1000 for anti-TH, Millipore). Sections were then washed in PBST and incubated for 1 hr at room temperature with the corresponding Cy- (1:200, Jackson Immunoresearch) or Alexa Fluor-conjugated secondary antibodies (1:500, Thermo Fisher Scientific) in 1% NDS-PBST. Sections were finally washed in PBS and rinsed in water before mounting onto slides with Fluoromount G (Electron Microscopy Sciences) for microscopic analysis.

Cultured epithelial cells and primary EGCs were grown to confluence on coverslips or transwell filter chambers (0.4µm pore size, Falcon, Heidelberg, Germany). After incubation with or without different mediators or supernatants for 24 h, cells were fixed with 2% formaldehyde for 10 min and then permeabilized with 0.1% Triton-X for 15 min at room temperature. Monolayers were next incubated at 4 °C overnight with mouse anti-Desmoglein2 (Thermo Fisher Scientific, Waltham, MA, USA) and mouse anti-E-Cadherin (BD Biosciences, Franklin Lakes, USA) diluted 1:100 with phosphate-buffered saline (PBS) or rabbit anti-Claudin1 and rabbit anti-Claudin5 (Both Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:50 with PBS, as well as chicken anti GFAP (Abcam, Cambridge, UK) diluted 1:60 in PBS. We used Cy3- or 488- labeled goat anti-mouse or goat anti-rabbit (all diluted 1:600, Dianova, Hamburg, Germany) and donkey anti-chicken FITC (Biozol, Eching, Germany as secondary antibodies. Coverslips and filters were placed on glass slides. To counterstain cell nuclei Vector Shield Mounting Medium as an anti-fading compound, which included DAPI (Vector Laboratories, Burlingham, CA, USA) was used. Representative experiments were photographed with a confocal microscope (Leica TCS SP2, Wetzlar, Germany).