Ab52636

For Western blot, the procedures of electrophoresis, transfer, and immunodetection were performed according to our previous study (Xu et al., 2012 (link); Hu et al., 2013 (link)). The primary antibodies used were as follows: antibody for the Ras-related protein Rab-3A (Rab3a, ab3335, 1:2000); syntaxin-binding protein 1 (Stxbp1, Munc18-1, ab124920, 1:4000); synapsin-1 (Syn1, ab18814, 1:1000); syntaxin-1A (Stx1a, ab41453, 1:3000); synaptosomal-associated protein 25 (SNAP25, ab5666, 1:4000); vesicle-associated membrane protein 2 (VAMP2, ab3347, 1:2000); synaptophysin (ab52636, 1:1000) (all purchased from Abcam); synaptotagmin 1 (Syt1, Millipore MAB5200, 1:1000); syntaxin-1B (Stx1b, Synaptic Systems 110402, 1:1000); and PSD95 (CST #3450, 1:1000). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG (purchased from Bio-Rad, dilution 1:15000) were used as secondary antibodies. After immunodetection, the intensity of the immunostained bands were normalized for the total protein intensities measured by Coomassie blue from the same blot (Van den Oever et al., 2008 (link); Counotte et al., 2010 (link)). The images were subjected to densitometric analysis performed using Quantity One software (Bio-Rad).

Phosphatase inhibitors and protease inhibitors (Millipore, Burlington, MA, USA) were added to SDS lysis buffers to homogenize the hippocampus and cortex. With the Bradford method (Bio-Rad, Hercules, CA, USA), the protein supernatants of the lysates were obtained after centrifugation at 15,000 r and 4 °C for 15 min. Proteins were transferred to PVDF membranes (Millipore) after isolation on SDS-PAGE gels, sealed with skim milk and detected overnight with primary antibody at 4 °C. Overnight incubation occurred of endoplasmic reticulum stress related antibodies, against apoptotic antibodies and neuroinflammation antibodies: PSD95 antibody #ab12093 (abcam, Cambridge, UK); Synaptophysin antibody #ab52636 (abcam); GFAP antibody #95717 (Cell Signaling Technology(CST), Boston, MA, USA); Anti-TNF Receptor I antibody #ab19139 (abcam); Bax antibody #AF0120 (Affinity Biosciences, Cincinnati, OH, USA); Bcl-2 antibody #AF6139 (Affinity Biosciences); β-actin antibody # A1978 (Sigma, Ronkonkoma, NY, USA); Anti-rabbit IgG (CST). Immunoreaction of anti-mouse IgG antibody (CST) was completed, ECL luminescent solution was 1:1, and the immunoblotting was recorded by ChemiDocXRS+ (Bio-RAD, Hercules, CA, USA), a comprehensive gel imager.

IHC for AR (ab9474, Abcam), FGFR1 (9740, CST), P-ERK (4370, CST), SOX2 (3579, CST), CHGA (ab45179, Abcam), SYP (ab52636, Abcam) and ARHGEF2 (ab155785, Abcam) was performed using PV-6000 system (ZSGB-BIO, China). Briefly, the slides were immersed in 1X Tris-EDTA (pH 9.0) buffer (Solarbio, China) and placed in a microwave oven for 10 min on high heat, and then adjusted to medium-low heat for 10 min to restore the antigen. 3% H₂O₂ was added to remove endogenous peroxidase in tissue samples. Cover the tissue on the slide with the primary antibody, place it in a humid box, and incubate overnight at 4 °C. After rewarming at room temperature for 30 min, horseradish peroxidase-linked secondary antibody (ZSGB-BIO, PV-6000, China) was added to the specimen and incubate the slides at room temperature for 30 min. After being stained with the DAB solution, the slides were immediately placed in water to stop dyeing, slides were subsequently counterstained with hematoxylin. The tissue is then dehydrated and preserved with neutral balsam (OriGene, ZLI- 9555, China).

SH-SY5Y cells were treated by RIPA Lysis Buffer (Epizyme, China) with 1 mM PMSF (Beyotime, China) on ice for 5 s. Then, the lysate was centrifuged at 14,000 g for 5 min. The supernatant was collected and the concentration of total protein was quantified by bicinchoninic acid assay (Beyotime, China). 15 μg of proteins were loaded and separated on 10% SDS-PAGE. Then, proteins were transferred onto PVDF membranes which were blocked with 5% non-fat milk in TBST buffer at room temperature for 1 h. The membranes were incubated with appropriate primary antibodies at 4°C overnight and then with corresponding secondary antibodies. The optical density of protein bands was quantified using Image J. Antibodies used in experiments were as follows: REST (Abcam, #ab75785), ARC (Proteintech, #16290-1-AP), synaptophysin (Abcam, #ab52636), PSD-95 (Abcam, #ab18258), GAPDH (BBI, #D110016), TUBULIN (Affinity, #AF7011), goat anti-rabbit IgG H&L (Abcam, #ab6721), and goat anti-Mouse IgG H&L (Abcam, #ab205719).

Equal amounts of protein (30 μg) were loaded onto 10% (W/V) sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoresed. The proteins were transferred onto PVDF membrane (Millipore) and incubated with the primary antibodies of MEGF10 (1:700, A10508, ABclonal, MA), MERTK (1:700, AF591, R&D, MN), SYP (1:800, ab52636, Abcam), Homer-1 (1:800, ab184955, Abcam) and β-actin (1:1000, MA5–15739, Invitrogen) at 4 °C overnight. The membrane was washed in TBST buffer and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (1:5000, Invitrogen) for 1 hr at RT, and then reacted with an enhanced chemiluminescence substrate (Meilunbio, Shanghai, China). The result of chemiluminescence was recorded and semi-quantified using the ImageJ software (NIH, Bethesda, MD). Bright-field image and chemiluminescent blots are merged using Tanon GIS software (www.Bio-tanon.com.cn).

IHC for AR (ab9474, Abcam), FGFR1 (9740, CST), P-ERK (4370, CST), SOX2 (3579, CST), CHGA (ab45179, Abcam), SYP (ab52636, Abcam) and ARHGEF2 (ab155785, Abcam) was performed using PV-6000 system (ZSGB-BIO, China). Briefly, the slides were immersed in 1X Tris-EDTA (pH 9.0) buffer (Solarbio, China) and placed in a microwave oven for 10 min on high heat, and then adjusted to medium-low heat for 10 min to restore the antigen. 3% H₂O₂ was added to remove endogenous peroxidase in tissue samples. Cover the tissue on the slide with the primary antibody, place it in a humid box, and incubate overnight at 4 °C. After rewarming at room temperature for 30 min, horseradish peroxidase-linked secondary antibody (ZSGB-BIO, PV-6000, China) was added to the specimen and incubate the slides at room temperature for 30 min. After being stained with the DAB solution, the slides were immediately placed in water to stop dyeing, slides were subsequently counterstained with hematoxylin. The tissue is then dehydrated and preserved with neutral balsam (OriGene, ZLI- 9555, China).

Infarcted hemispheres were homogenized and lysed using RIPA lysis buffer (G2002, Servicebio, Wuhan, China), supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche), followed by sonication on ice. The supernatant was collected after centrifugation (12,000 g, 30 min). Protein concentration was quantified using a BCA reagent (G2026, Servicebio, Wuhan, China). Western blot procedures were based on previous literature.
¹⁴ (link) The primary antibodies used in our study comprised the following: anti‐annexin A6 rabbit polyclonal antibody (1:2000 dilution, 12,542‐1‐AP, Proteintech), anti‐neuroligin rabbit polyclonal antibody (1:1000 dilution, ab36602, Abcam), anti‐myelin basic protein (MBP) rabbit polyclonal antibody (1:1000 dilution, ab40390, Abcam), anti‐TrkB rabbit polyclonal antibody (1:1000 dilution, ab18987, Abcam), anti‐synaptophysin rabbit monoclonal antibody (1:5000 dilution, ab52636, Abcam), and anti‐β‐actin mouse monoclonal antibody (1:2000 dilution, GB15001‐100, Servicebio). The appropriate secondary antibodies used were horseradish peroxidase‐conjugated goat anti‐rabbit (1:2500 dilution, ZB‐2301, ZSGB‐BIO, Beijing, China) or goat anti‐mouse antibody (1:2500 dilution, ZB‐2305, ZSGB‐BIO, Beijing, China).