Mouse neun

Twenty μm thick coronal sections were washed with PBS and 0.4% Triton-X PBS for 20 minutes. The sections were then blocked with 10% normal donkey serum for 1 hour at room temperature in PBS containing 0.1% Triton X-100, followed by incubation with primary antibody for 1–3 nights at 4°C in the same buffer. Primary antibodies used for this study included rabbit-NLRP3 (Santa-Cruz Biotechnology, sc-66846), rabbit-ASC (Santa-Cruz Biotechnology, sc-22514-R), goat-cleaved caspase-1 (Santa-Cruz Biotechnology, sc-22165), rabbit-IL-1β (Abcam, ab9722), goat-Iba1 (Abcam, ab5076), rabbit-P2X7 receptor (Sigma, P8232), and mouse-NeuN (Millipore, MAB377). After primary antibody incubation, sections were washed for 3 × 10 minutes at room temperature (RT), followed by incubation with the appropriate secondary antibody: Alexa-Fluor488/568/647 donkey anti-rabbit/anti-mouse/anti-goat (Invitrogen) RT/1 hour. Sections were then washed with PBS containing 0.1% Triton X-100 for 3 × 10 min, followed by 2 × 5 min with 1x PBS and briefly with water. The sections were then mounted with water-based mounting medium containing antifading agents and observed using confocal microscopy. All images were captured on a confocal laser microscope (Carl Zeiss, Germany) using the Zen software at 40x magnification and 50 μm scale bar.

At the end of both IEG tests, subjects were immediately euthanized by isoflurane overdose and were transcardially perfused with 0.1 M phosphate buffer saline (PBS) followed by 4% paraformaldehyde. Brains were extracted, post-fixed overnight in 4% paraformaldehyde, and underwent cryoprotection in 30% sucrose dissolved in PBS for 48 h. Brains were frozen in Tissue-Tek O.C.T. compound and stored at − 80 °C before sectioning coronally at 40 µm using a Leica cryostat, with every third section saved for use in the present study. Tissue sections were immunofluorescently stained for Fos (the protein of the immediate early gene cFos; Synaptic Systems rabbit c-fos 1:1000 dilution) and NeuN, a neuron-specific nucleus marker (Millipore mouse NeuN 2:1000 dilution).

Cells were fixed with 4% paraformaldehyde, and rinsed twice with 1× PBS. Primary antibodies included rabbit CD86 (1 μl/ml; Abcam, Cambridge, MA), rat CD206 (5 μl/ml; Abcam), rabbit Beta III Tublin (5 μl/ml, Abcam), rabbit anti-cleaved caspase-3 (0.5 μl/ml; Abcam), and mouse NeuN (10 μl/ml; Millipore, Billerica, MA). In addition, cell nuclei were stained with DAPI (0.5 μl/ml; Life Technologies, Carlsbad, CA). Fluorescently tagged secondary antibodies (Invitrogen) were visualized with an Olympus BX43 fluorescent microscope with a CellSens Standard imaging program. Five randomly selected images were taken per sample and each image was qualitatively assessed for overall fluorescence. NeuN-positive cells were manually counted using NIH ImageJ (Bethesda, MD).

Primary antibodies used in the study were rabbit Calbindin D28-k (Swant, CB-38, 1:5,000), rat DA transporter DAT (Millipore, MAB369, 1:1,000), rabbit glial fibrillary acidic protein GFAP (DAKO, ZO334, 1:1,000), chicken GFP (Abcam, ab13970, 1:500), rabbit Iba1 (WAKO, 019–19741, 1:1,000), mouse NeuN (Millipore, MAB377, 1:300), rat anti-mouse CD49d, Clone R1-2 (RUO) (BD Horizon, 1:400), mouse tyrosine hydroxylase TH (Millipore, MAB318, 1:1,000), rat anti-mouse CD31 (BD Pharmingen 557355, 1:100), and mouse Olig2 (Millipore MABN50, clone 211F1.1, 1:100).
Secondary antibodies used were Alexa Fluor 647 goat anti-rat (Life Technologies, A21247, 1:1,000; Carlsbad, CA, USA), Alexa Fluor 488 goat anti-chicken (Abcam, ab150169, 1:1,000), Alexa Fluor 568 goat anti-mouse (Invitrogen, A11004, 1:1,000; Carlsbad, CA, USA), Alexa Fluor 647 goat anti-mouse (Invitrogen, A21235, 1:1,000), Alexa Fluor 568 goat anti-rabbit (Invitrogen, A11011, 1:1,000), and Alexa Fluor 647 goat anti-rabbit (Invitrogen, A21244, 1:1,000).

Except where indicated, staining was done on 40-μm free-floating sections of a one-in-six series through the entire hippocampus and cortex. Tissue was blocked in 10 % normal serum from the species in which the secondary antibody was raised, with the addition of 0.1 % Triton X-100. Primary antibodies were diluted in 3 % normal serum with 0.1 % Triton X-100, and tissue incubated overnight at 4 °C. Biotinylated secondary antibodies were diluted in 3 % normal serum with 0.1 % Triton X-100 and were incubated for 2 h at room temperature. Enzyme detection was done using avidin-biotin substrate (ABC kit, Vector Laboratories, Burlingame, CA, USA) followed by color development in diaminobenzidine solution (Sigma-Aldrich, St. Louis, MO, USA). Antibodies and dilutions were as follows: rat CD11b (Serotec, Raleigh, NC, USA; 1:500); mouse CD68 (Serotec; 1:200); rabbit YM1 (Stem cell technology, Vancouver, BC, Canada; 1:400); rat Marco (Serotec; 1:500); mouse NeuN (Millipore, Temecula, CA, USA; 1:1000). After incubation for 24–48 h with appropriate primary and biotinylated secondary antibodies, tissue was treated with Vectastain ABC reagent (Vector Labs) and visualized with DAB reaction (Sigma-Aldrich). Controls included omitting primary or secondary  antibodies.

The following primary antibodies were used: mouse NeuN (1:500; Millipore); mouse Calbindin (1:1000; SWant); mouse Calretinin (1:250; BD Transduction Laboratories) rabbit Prox1 (1:500; Millipore); rabbit DCX (1:250; Santa Cruz); mouse NF-pan (1:3; Zymed); rabbit GFP (1:1000; Life Technologies). To detect primary antibodies, the following appropriate species-specific secondary antibodies were used: Goat anti-rabbit Alexa-488 (1:500; Life Technologies), Goat anti-mouse Alexa-568 (1:500; Life Technologies).