Rabbit anti gfap

We visualized NLRP3 inflammasomes in microglia, neurons, and astrocytes through triple immune-fluorescence staining of brain tissue sections (n=6/group) for NLRP3, an apoptosis-associated speck-like protein containing a CARD (ASC), and IBA-1, NeuN, or GFAP. Goat anti-NLRP3 (1:500, Millipore), mouse anti-ASC (1:1000, Santa Cruz), rabbit anti-NeuN (1:1000, Millipore), rabbit anti-GFAP (1:1000, Millipore), rabbit anti-iba1 (1:1000 Abcam), donkey anti-goat IgG tagged Alexa Fluor 488 (1:200, Invitrogen), donkey anti-mouse Alexa Fluor 594 (1:200, Invitrogen), and donkey anti-rabbit 405 (1:200, Invitrogen) were employed in these studies. Two-micrometer thick optical Z-sections were employed for blinded quantification of the total number of NLRP3 inflammasomes (i.e., structures positive for both NLRP3 and ASC) in 216 μm² area of the CA3 subfield of the hippocampus (2 sections/animal, n=5-6/group). We also quantified the percentages of IBA-1+ microglia, NeuN+ neurons, and GFAP+ astrocytes containing NLRP3 inflammasomes (2 sections/animal) [25 (link)].

Total protein was extracted with cell lysis buffer (Solarbio, Beijing, China). Proteins were separated by 10% SDS‐PAGE and then transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 5% non‐fat milk (Sangon Biotech, Shanghai, China) for 2 h, then incubated with primary antibodies overnight at 4°C, and finally incubated with HRP‐conjugated secondary antibodies for 2 h at room temperature. Next, immunoreactive bands were detected using an enhanced chemiluminescence kit (Bio‐Rad, Hercules, CA, USA). β‐Actin was used as a loading control. The primary antibodies included mouse anti‐MAP2 (Millipore; 1:1000), mouse anti‐Tuj1 (Millipore; 1:1000), rabbit anti‐GFAP (Millipore, 1:1000), rabbit anti‐Acsl4 (Abcam; 1:1000), rabbit anti‐Akt (Cell Signaling Technology, Danvers, MA, USA; 1:1000), rabbit anti‐phospho‐Akt (Cell Signaling Technology; 1:1,000), rabbit anti‐PI3K (Abcam; 1:1000) and mouse anti‐β‐actin (Abcam; 1:1000).

Before using hydrogels in vivo, their biocompatibility with VM cultures was assessed. VM cultures were incubated with preformed collagen hydrogels (2 × 50 µl gels) cross-linked with 1, 2 or 4 mg/ml of cross-linker for 24 h or left untreated. Following incubation, cells were either assessed for cell viability using the AlamarBlue® (Invitrogen) assay or fixed for future immunocytochemical staining for neurons (using mouse anti-β-III tubulin from Millipore @ 1:200), dopaminergic neurons (using mouse anti-tyrosine hydroxylase from Millipore @ 1:1000) or astrocytes (using rabbit anti-GFAP from Millipore @ 1:2000, respectively). For analysis, cell counts were quantified from five randomly selected sample sites per well, in three technical replicates per experimental condition, with three biological replicates. The β-III tubulin florescence was quantified by measuring the threshold area of each image using ImageJ software.

The BM MSC and neurosphere phenotypes were determined by immunological labelling. Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature and blocked with a solution containing 5% of bovine serum albumin in PBS and 0.05% Triton X-100 for cell permeabilization. Immunocytochemistry was carried out according to standard protocols. Cell nuclei were counterstained with SYTOX® Blue nucleic acid stain (1:1000; Invitrogen, USA). The antibodies used and dilution were as follows: Anti-CD90 (FITC) conjugated mouse monoclonal antibody (1:200; Thermo Scientific, USA); Anti-CD44 (PE) conjugated rat monoclonal antibody (1:200; Abcam, Cambridge, UK) [17 (link)]; Alexa Fluor® 647 mouse anti-nestin (1:200; BD Pharmingen, USA); Milli-Mark™ FluoroPan neuronal marker (1:100; Catalog # MAB2300X, Millipore, USA); Anti-Sox-2 clone 6G1.2 FITC conjugated antibody (1:100; Millipore, USA); Rabbit anti-GFAP (1:100; Millipore, USA) and donkey anti-rabbit IgG-FITC secondary antibody (1:250; Santa Cruz Biotechnology, Inc, USA). UltraCruz™ anti-fading mounting medium (Catalog # Sc-24941, Santa Cruz Biotechnology, Inc, USA) was applied to minimise photo bleaching due to exposure to high-intensity light. The fixed cells were then viewed using a Pascal 5 confocal microscope (Carl Zeiss, Germany).

The following antibodies were used: mouse anti-Tuj1 (1:50, Millipore), mouse anti-microtubule-associated protein (MAP) 2 (1:200; Sigma–Aldrich), rabbit anti-GFAP (1:200; Millipore), mouse anti-O4 (1:100, Millipore), mouse anti-CNPase (1:100, Millipore), goat anti-Sox2 (1:100, Santa Cruz), mouse anti-Nestin (1:100; Developmental Studies Hybridoma Bank, Beijing, China), mouse anti-Pax6 (1:100; Developmental Studies Hybridoma Bank), donkey Alexa-555 anti-mouse (1:1000; Invitrogen), and donkey Alexa-555 anti-rabbit (1:1000; Invitrogen). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The slide-mounted, stained samples were observed using an LSM 510 META confocal microscope (Zeiss, Oberkochen, Germany) with excitation wavelengths of 543, 488, and 405 nm. The channel signals were collected sequentially. Collected images were assembled using Adobe Photoshop (Adobe Systems, San Jose, CA).

Mouse brains were fixed in 4% PFA at 4 °C overnight and cryosectioned at a thickness of 30 µm. The sections were rehydrated, blocked by 10% goat serum, and incubated with primary antibodies: mouse anti-tyrosine hydroxylase (1:1000, Sigma), mouse anti-nestin (1:500, Abcam), rabbit anti-Sox2 (1:500, Abcam), rabbit anti-Iba1 (1:500, Wako), rabbit anti-GFAP (1:1000, Millipore), and rabbit anti-BrdU (1:500, Abcam). For immunohistochemistry staining, the sections were washed and incubated with biotinylated anti-mouse or anti-rabbit secondary antibodies (1:200, Vector Laboratories, USA), and then with AB peroxidase (1:200, Vector Laboratories, USA). The peroxidase reaction was detected with 0.05% DAB (Sigma, USA) in 0.1 M Tris-HCl buffer and 0.03% H₂O₂. For immunofluorescence staining, the sections were then washed and labeled with fluorescent secondary antibodies (1:500, Invitrogen). Sections were mounted with ProLong Gold Antifade Reagent with DAPI (1:5000, Invitrogen), and images taken under a Leica confocal microscope (TCS SP-2, Leica, Germany).