Ab13552

For double-immunofluorescence of TRPV1 and PSD95 (a marker for post synaptic mechanisms), free-floating sections containing PFC were washed in PBS and then preincubated in PBS containing 5% normal goat serum for 3–4 h at RT. After preincubation, sections were incubated with polyclonal rabbit anti-TRPV1 antibody (ab31895, Abcam, dilution: 1:500), and mouse monoclonal antibody anti-PSD95 (ab13552; Abcam; 1:500) for 24 h at 4°C, after which they were rinsed 3–5 times in PBS, the sections were then incubated for 1–2 h at RT in goat anti-rabbit IgG Alexa Fluor 488 (ab150077; Invitrogen; 1:1500) and goat anti mouse IgG Alexa Fluor 647 (ab150115; Abcam; 1:1500) dissolved in blocking solution. Following the final rinsing, the slices were wet mounted onto subbed slides and subsequently dried and cover-slipped with Dako Faramount Aqueous Mounting Medium (Dako; S3025). For fluorescence imaging, tissues were visualized under ab epifluorescence IX81 microscope (Olympus), and for confocal imaging a 700-AX10 laser scanning microscope (Carl Zeiss) was used.

The mouse hippocampus was lysed by radioimmunoprecipitation lysis buffer (Solarbio, Beijing, China, R0010) with a protease phosphatase inhibitor mixture (Solarbio, P1260). A BCA kit (Solarbio, PC0020) was applied to quantify the total protein concentration. Protein samples (40 μg per lane) were separated by 10% SDS-PAGE (Solarbio, P1200) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA, IPFL00010). The membranes were blocked with 5% skim milk (Solarbio, D8340) at room temperature (1 h) and subsequently immunoblotted with primary antibodies overnight at 4 °C: IL-33 (R&D, MND, USA, 1:500, AF3626), glial fibrillary acidic protein (GFAP) (Abcam, Cambridge, UK, 1:1000, ab7260), vesicular glutamate transporter 1 (vGlut1) (Abcam, 1:1000, ab227805), postsynaptic density protein-95 (PSD95) (Abcam, 1:1000, ab13552) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, 1:2000, ab8245). Next, 1X tris-buffered saline Tween (TBST) (Solarbio, T1081) was used to wash the membranes 3 times. They were then incubated with a secondary antibody at room temperature (40 min). Immunoreactivity was detected using enhanced chemiluminescence (Solarbio, P0018), and bands were measured using Image J analysis software (Version 1.50i, Rockville, MD, USA).

Mice were sacrificed under deep anaesthesia with 1% pentobarbital immediately after the final behavioural test. The entire tissue of the left V1 was lysed in RIPA lysis buffer and collected after centrifugation at 12,000 rpm for 15 min. Quantification of protein in supernatants was performed using the BCA assay (Thermo Fisher Scientific). Equal amounts of protein extracts were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). Membranes were blocked and probed with following appropriate antibodies according to standard procedures: ABCA1 (1:1,000, Abcam, ab7360), ApoA1 (1:1,000, Invitrogen, MIA1404), PSD-95 (1:1,000, Abcam, ab13552), Synapsin I (1:1,000, Abcam, ab64581), Synaptophysin (1:20,000, Abcam, ab32127) and β-Actin (1:1,000, Abcam, ab8226). After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature, the bands were visualised using enhanced chemiluminescence (ECL) according to the manufacturer’s protocols. Protein quantification was obtained using ImageJ software (http://imagej.net/ImageJ) and the specific protein expression level was normalised to the level of β-actin on the same PVDF membrane.

All drugs and reagents were from Sigma unless otherwise indicated. The main reagents were as follows: Neurobasal medium (Gibco), B27 Supplement (Gibco), Sulfo-NHS-LC-Biotin (Thermo Scientific), NeutrAvidin Agarose Resins (Thermo Scientific), recombinant human BDNF (R&D System), and K252a (Tocris).
The antibodies used for western blotting were ASIC1a (1:500, sc-13905, Santa Cruz) and GAPDH (1:2000, KC-5G4, KangChen). The HRP-conjugated secondary antibodies used for western blotting were goat anti-rabbit IgG (1:2000, AP132P, Millipore), rabbit anti-mouse IgG (1:2000, AP160P, Millipore), and rabbit anti-goat IgG (1:2000, AP106P, Millipore). The primary antibodies used for immunostaining were GFP (1:1000, A10262, Invitrogen), HA (1:1000, 901501, BioLegend), DsRed (1:1000, 632496, Clontech), and PSD-95 (1:1000, ab13552, Abcam). The secondary antibodies used for immunostaining were donkey anti-human IgG DyLight 550 (1:1000, SA5-10127, ThermoFisher), donkey anti-human IgG DyLight 488 (1:1000, SA5-10126, ThermoFisher), donkey anti-mouse IgG Alexa 647 (1:1000, 715-605-150, Jackson ImmunoResearch), donkey anti-rabbit IgG Alexa 568 (1:1000, A10042, Invitrogen), and goat anti-chicken IgG Alexa 488 (1:1000, A11039, Invitrogen).

Brains were collected and fixed overnight with 4% PFA in PBS at 4°C and dehydrated in 30% sucrose in PBS at 4°C. For sections or isolated cells, cryo‐sections (40 μm thick) or coverslips were fixed with 4% PFA/PBS, and permeabilized with 0.5% Triton X‐100 for 10 min and blocked in blocking buffer (0.3% Triton X‐100, 2% Bovine Serum Albumin) for 1 h at room temperature. Primary antibodies were incubated at 4°C overnight. The primary antibodies used in this study are as follows: rabbit anti‐Arid1a (1:1,000, HPA005456, Sigma), pig anti‐Vgult1 (1:1,000, ab5905, Millipore), rabbit anti‐H3K27ac (1:1,000, ab4729, Abcam), chick anti‐MAP2 (1:1,000, Cat No.822501, Biolegend), rabbit anti‐PSD95(1:1,000, ab16659, Abcam), mouse anti‐synaptophysin (1:1,000, ab13552, Abcam), mouse anti‐Oct‐3/4 (1;1,000, Cat No. sc‐5,279, Santa Cruz), rabbit anti‐Nanog (1:1,000, Cat No. 14295–1, Proteintech), and rat anti‐BrdU (1:1,000, Cat No. ab6326, Abcam). Sections or coverslips were washed in PBS and incubated with secondary Alexa Fluor‐conjugated antibodies (Invitrogen) for 1.5 h at room temperature, washed three times in PBS, and mounted with an adhesion antifade medium. We used a confocal laser‐scanning microscope to obtain images and ImageJ to analyze the results.