Mouse anti map2

Human SH-SY5Y neuroblastoma cells were transfected with target siRNAs for 24 h and then treated with retinoic acid for 48 h. Cells were then fixed in 4% paraformaldehyde for 1 h at room temperature (RT), permeabilized with 0.1% Triton X-100 in PBS for 15 min, and incubated in blocking reagent (5% normal fetal bovine serum in PBS) for 1 h. Cells were incubated with primary antibody against MAP2, a neuronal marker (Mouse Anti-MAP2, Abcam, Cambridge, United Kingdom), at 4°C overnight, followed by incubation with a secondary antibody (Alexa Fluor 488, Goat anti-mouse, Life Technologies, California, United States) at RT for 1 h. For nuclear counterstaining, the cells were incubated with DAPI solution (300 nM in PBS) for 5 min at RT and then observed with a fluorescence microscope (Eclipse Ti-S, Nikon, Tokyo, Japan). To measure neurite length in SH-SY5Y cells, neurite length was calculated as the longest neurite distance from the cell body (direct path to the soma) on each neuron showing MAP2 (green) using ImageJ software (10 calculated cells per group). The mRNA expression levels of target genes and MAP2 were analyzed by real-time PCR.

The following primary antibodies were used for immunocytostaining: rabbit anti-S100β (dilution = 1 : 200) (Abcam, Cambridge, UK), rabbit anti-GAP43 (1 : 200) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-EGR2 (1 : 200) (Abcam), rabbit anti-NCAM (1 : 200) (Sino Biological, Beijing, China), and mouse anti-MAP-2 (1 : 200) (Abcam) antibodies. Alexa Fluor 488-conjugated anti-rabbit (1 : 500) (Life Technologies, Carlsbad, CA, USA) and Alexa Fluor 594-conjugated anti-mouse (1 : 500) (Life Technologies) antibodies were used as secondary antibodies. For western blotting, rabbit anti-Smad2/3 (dilution = 1 : 1000) (Cell Signaling Technology), rabbit anti-phospho Smad2/3 (1 : 5000) (Cell Signaling Technology), and rabbit anti-β-actin (1 : 5000) (Cell Signaling Technology) antibodies were used. For flow cytometric analysis, phycoerythrin- (PE-) conjugated mouse anti-human CD29 (dilution = 1 : 25) (BioLegend, San Diego, CA, USA), Alexa Fluor 488-conjugated rat anti-mouse/human CD44 (1 : 25) (BioLegend), PE-conjugated mouse anti-human CD90 (1 : 5) (BD Biosciences, San Jose, CA, USA), and fluorescein isothiocyanate- (FITC-) conjugated mouse anti-human CD105 (1 : 25) (BioLegend) antibodies were used.

Brain tissues were fixed in 4% paraformaldehyde and then embedded in paraffin. Then, 5 μm thick sections of brain tissues were deparaffinized with xylene and blocked with goat serum. For primary cortical neurons, cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with goat serum. Prepared samples were incubated with primary antibodies of rabbit anti-α7nAChR (1:200, Proteintech, Wuhan, Hubei, PRC) with or without mouse anti-MAP2 (1:10,000, Abcam, Cambridge, MA, United States) and rabbit anti-LC3 (1:50, Proteintech, Wuhan, Hubei, PRC) with mouse anti-MAP2. After that, sections and dishes were incubated with double immunofluorescent staining including Alexa-488 and Alexa-647 labeled secondary antibodies (1:500, Invitrogen, United States) for 1 h at room temperature. DAPI (Vector Laboratories, Burlingame, CA, United States) was used to stain the nucleus. The sections and dishes were scanned by using a confocal laser scanning microscope (FluoView FV1000; Olympus, Tokyo, Japan). To perform semi-quantitative statistical analysis, the average fluorescence intensity was detected with Image J software. In brief, we split a single channel of the image and then adjusted the threshold with the default method. After setting measurements, we can get the values.

The LPS ± NAC treated cortical cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, followed by PBS washes. The fixed neurons were then immunostained with mouse anti-MAP2 (neuron marker) (1:500, Abcam, Cambridge, MA). MAP2 positive neurons were then visualized with FITC or Alexa Fluor 488 anti-mouse secondary antibodies (Jackson ImmunoResearch Inc.) under a 20X objective. Nuclei were stained using DAPI (Sigma) to determine cell survival. At least twenty different objective views were randomly selected from two to three independent experiments. The experimenter was blinded to the condition when taking images and counting. Neurons with positive MAP2 immunostaining, and even and intact DAPI staining were considered alive.

STORM was performed using a Nikon A1 confocal microscope with CFI SR Apochromat TIRF ×100 oil objective and or Technology iXon3 897 EMCCD camera (Nikon Instruments Inc., Melville, NY, USA). Samples were treated according to the manufacturer’s protocol as previously described^{15 (link)}. Rabbit anti-Tom20 (1:25; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), mouse anti-Drp1 (1:1000; Abcam), and mouse anti-MAP2 (1:1000; Abcam) were used overnight to label mitochondria, Drp1, and neuronal cytoskeleton, respectively.

Spinal cords were removed from 4-week post trauma rats and fixed in 4% paraformaldehyde for 30 min. A 3-mm long section of the spinal cord, centered on the injured area, was cut into 35-μm-thick sections using a Leica RM2135 microtome. The sections were prepared for immunohistochemistry as described in our previous studiy [31 (link)]. The primary antibodies used included mouse anti-Map-2 for neurons (1:500; Abcam, UK) and rabbit anti-glial fibrillary acidic protein (GFAP) for astroglia (1:1000; Abcam, UK); the secondary antibodies used were Alexa Fluor 488 (green, 1:1000; Molecular Probes, Germany) and Cy5 (red, 1:500; Dianova, Germany). The sections were observed and photographed using a DM-6B fluorescence microscope (Leica, Germany) connected to a computer screen.