Ab254349

At 3 days, 2 weeks, and 2 months after sub-FS stimuli, rats were anesthetized and decapitated. Brains were quickly removed to separate the hippocampus and cortex on ice. After measuring protein content using a BCA Protein Assay Kit (P0012; Beyotime), proteins were separated using 10% and 12% SDS-PAGE gel (P0012AC; Beyotime) and transferred to a polyvinylidene fluoride membrane (220 mA, 60 or 100 min, respectively). Subsequently, these membranes were blocked with 5% skim milk for 3 h. After incubation with the primary antibodies anti-synapsin I (1:1 000; ab254349; Abcam), anti-PSD95 (1:1 000; ab2723; Abcam), anti-VGLUT1 (1:1 000; ab227805; Abcam), anti-TGF-β1 (1:1 000; ab179695; Abcam), anti-TSP-1 (1:1 000; A00667-1; Boster), anti-caspase-3 (1:1 000; 9662; Cell Signaling Technology), anti-cleaved caspase-3 (1:1 000; ab2302; Abcam), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1 000; AB-P-R-001; Goodhere) overnight at 4°C, and the horseradish peroxidase-conjugated secondary antibody (1:3 000; ZB-2301 and ZB-2305; Beijing Zhongshan) for 2 h at 37°C, the membranes were exposed (ImageQuant LAS 500, USA). Gel bands were analyzed using ImageJ (version 1.49; National Institutes of Health; United States) software and are presented as ratios to GAPDH.

Cells were washed three times with sterile PBS and then fixed using 4% PFA for 20 min. After washing, cells were blocked 0.3% Triton-X and 1% goat serum in PBS for 1 h. Primary antibodies specific for Synapsin1 (1:500; ab254349; Rabbit; Abcam, Cambridge, MA, USA), NeuN (1:500; ab104225; Rabbit; Abcam, Cambridge, MA, USA), Beta-III Tubulin (1:500; MAB1637, Mouse; Kenilworth, NJ, USA), MAP2 (1:1000; Chicken; ab5392; Abcam, Cambridge, MA, USA), TBR1 (1:200; ab183032; Rabbit; Abcam, Cambridge, MA, USA), GFAP (1:500; ab4674; Chicken; Abcam, Cambridge, MA, USA), and Ki67 (1:500; ab245113; Mouse; Abcam, Cambridge, MA, USA) were incubated overnight. After washing, secondary antibodies (chicken 555, rabbit 488, mouse 647; Abcam, Cambridge, MA, USA) were incubated for 2 h. This was followed by 10 min of DAPI Staining Solution in PBS (1:1000, ab228549, Abcam, Cambridge, MA, USA) after which point slides were cover-slipped with ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) mounting media and allowed to dry for 48 h.

Induced cardiomyocytes and sympathetic neurons from hiPSC were dissociated and seeded on FluoroDish tissue culture dishes at desirable density for subsequent confocal imaging. Two to five days after seeding, cells were fixed with 4% formaldehyde, permeabilized and blocked in Tris-buffered saline (TBS) plus 0.5% Triton and 6% donkey serum. hiPSC-CM were then incubated in TBS with primary antibodies, anti-α-actinin (Sigma, A7811) and anti-cardiac troponin T (anti-cTnT, Abcam, ab45932), overnight at 4°C. For hiPSC-SN, anti-tyrosine hydroxylase (anti-TH, Sigma, T2928), anti-peripherin (anti-PRPH, Sigma, AB1530) and anti-Synapsin (anti-Syn, Abcam, ab254349) were used. Then secondary antibodies conjugated to Alexa Fluor (488 and 594) were applied at room temperature for 2 h. Cell nuclei were stained by Hoechst (Invitrogen, H3570) for 10 min. Images were taken by Leica confocal microscope (Leica TCS SP5) and analysed with LAS X software.

For immunostaining, brains were sliced into 50 μm coronal slices by a vibration microtome (Leica, VT1000 S). The brain slices were washed in PBS three times at room temperature, blocked with 5% (wt/vol) BSA (bovine serum albumin) containing 0.3% Triton-X-100 (vol/vol) in 0.01 M PBS for 1.5 h at room temperature and then incubated with primary antibodies for 24 h at 4 °C. The following primary antibodies were used: anti-PV (1:500, mouse, Millipore, MAB1572), anti-synaptophysin (1:200, mouse, Abcam, ab8049), anti-synapsin (1:500, rabbit, Abcam, ab254349), anti-SV2 (1:500, rabbit, Abcam, ab32942), anti-SNAP-25 (1:100, rabbit, Abcam, ab108990), anti-syntaxin (1:100, rabbit, Abcam, ab272736), anti-beta amyloid (1:800, mouse, Abcam, ab126649), and anti-GFP (1:500 rabbit, Abcam, ab290). Then, the sections were washed in PBS four times for 15 min and incubated with the corresponding secondary antibodies for 1.5 h at room temperature. All antibodies were diluted in the same block solution.