Cy3 conjugated

The detailed protocol of microarray-based serum screening was described by Li
et al. [
12 (link),
18 (link)]. Briefly, the protein microarray was blocked with 3% BSA buffer at 25°C for 3 h, and incubated with 200 μL diluted (1:200) serum at 25°C for 2 h. After 3 times wash with PBST, the microarray was incubated with anti-human IgG/IgM antibody at 25°C for 1 h with Cy3-conjugated and Alexa Fluor 647-conjugated, respectively (Jackson ImmunoResearch, Philadelphia, USA). After being washed with PBST for 3-times, the microarray was spin-dried at 25°C and scanned by LuxScan (CapitalBio, Beijing, China). The IgG and IgM signals were extracted with GenePix Pro 6.0 (Molecular Devices, San Jose, USA) and defined by foreground medians subtracted by background medians. The signal of a protein or peptide was averaged from triplicate spots.

For ex vivo immunofluorescence analysis, 8 or 11 week old mice, as indicated, were perfused with ice cold 0.1 M phosphate buffer followed by 4% paraformaldehyde. Brains were removed, post-fixed in 4% paraformaldehyde and 30% sucrose for 4 days before being frozen in Neg50 OCT (Thermo Scientific) 30 micron sections were taken and stored in PBS with sodium azide at 4 °C. Sections were incubated in 5% horse serum in 0.5% Triton-X TBS for 1 h before overnight incubation with 1:500 anti-GFP or 48 h 1:300 anti-UBE3A (3E5 Sigma) and 1 h 1:500 Alexa-Fluor 488 (Thermo Fisher Scientific, A-11039) or Cy3-conjugated (Jackson Immunoresearch, 715–165-151) secondary, respectively. Sections were stained with Hoechst for nuclei labeling before mounting in Fluoromount-G (Southern Biotech).

At the indicated timepoints after KA injection, mice (WT, Atf6b^−/−, and Calr^+/−) were deeply anesthetized with isoflurane and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde prepared in 0.1 M phosphate buffer (pH 7.4). Brains were harvested, post-fixed with 4% paraformaldehyde for 8 h, and cryoprotected in 30% sucrose for at least 24 h. Cortical Sects. (10 μm-thick coronal sections containing the hippocampus (between Bregma − 1.5 and − 2.1 mm)) were cut on a cryostat (Leica Biosystems, Wetzler, Germany). Sections were processed for Nissl staining (Cresyl violet staining) or immunohistochemistry with antibodies against cleaved caspase-3 (Asp175; Cell Signaling Technology, Inc. Danvers, MA, USA; 1:500) and c-Fos (PC05; Merck; 1:200). Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Anti-rabbit and anti-mouse Alexa Fluor 488-conjugated (Life Technologies; 1:200) and Cy3-conjugated (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 1:200) secondary antibodies were used to visualize immunolabeling. Imaging was performed using a laser scanning confocal microscope (Eclipse TE200U; Nikon, Tokyo, Japan) and Nikon EZ-C1 software or using a light and fluorescence microscope (BZ-X700; KEYENCE, Osaka, Japan).

According to our previous method (Fu et al., 2018 (link)), mice were anesthetized and transcardially perfused with saline followed by 4% paraformaldehyde (PFA). Subsequently, the brains were postfixed in 30% sucrose solution with 4% PFA. Serial coronal brain sections (30‐μm‐thick) were collected on a cryostat and preserved in a cryoprotectant at −20°C. The sections were incubated with the following primary antibodies (rabbit anti‐ionized calcium binding adapter molecule 1, Iba1, 1:1000, Wako) in 1% bovine serum albumin (BSA) overnight at 4°C, and 1% BSA replaced the primary antibody for the negative control. The next day, the sections were washed and incubated with Cy3‐conjugated (1:500, 2 h; Jackson ImmunoResearch, West Grove, PA, USA) secondary antibodies. Stained specimens were observed and captured using a Zeiss Axivert microscope (Oberkochen, Germany) equipped with a ZeissAxioCam digital color camera connected to the Zeiss AxioVision 3.0 system.
Microglia morphology was quantified according to a previous study (He et al., 2022 ). Eight‐bit 30 μm z‐stack images of Iba1⁺ cells were obtained with no more than a 2 μm interval between planes. Images were converted to binary by using Image J; Soma size, branch numbers, and branch length of microglia were measured using Image J with the plugin AnalyzeSkeleton.

Rats were perfused with 4% paraformaldehyde (PFA). The L4-6 DRGs were dissected and post-fixed in 4% PFA for 1 h. Then the tissues were dehydrated in 30% sucrose 3 days and embedded for cryostat sectioning. The cryostat sections (12 μm) were blocked with 3% donkey serum in 0.3% Triton X-100 for 1 h at room temperature and incubated in primary antibodies against Na_vβ1 (1:200, rabbit, Alomone Labs, Israel), Na_v1.8 (1:200, mouse, Abcam, United Kingdom) at 4°C overnight. After the primary antibody incubation, tissue sections were washed three times in 0.01 M PBS and then incubated in Cy3-conjugated (1:400, donkey anti-mouse, Jackson ImmunoResearch, United States) and FITC-conjugated (1:200, donkey anti-rabbit, Jackson ImmunoResearch, United States) secondary antibodies for 1 h at room temperature. The sections were rinsed with 0.01 M PBS three times and mounted on a coated slide, and air-dried. Three-dimensional super-resolution images were captured using a three-dimensional structured illumination microscope with the N-SIM System with an oil immersion objective lens CFI SR (Apochromat TIRF × 100, 1.49 numerical aperture, Nikon, Japan) and images were post-processed with Nikon NIS-Elements software.