Rabbit igg monoclonal isotype control

XN (Figure 1) was
provided by Yumen Tuopu Scientific and Technological Development Co., Ltd.,
purity > 98% (Yumen, Gansu, China). The cell culture medium RPMI-1640 was
purchased from Gibco Laboratories (Grand Island, NY, USA). Neonatal bovine serum
was obtained from Lanzhou Minhai Bio-engineering Co., Ltd. (Lanzhou, China).
Streptomycin, penicillin, L-glutamine, and dimethyl sulfoxide (DMSO) were all
greater than 98% in purity (Sigma-Aldirch Corp.). The
3-(4,5-dimethylthiazo-2-yl) −2,5 -diphenyl-tetrazolium bromide (MTT) kit and
Annexin V-FITC/PI apoptosis detection kit were obtained from KeyGEN Biotech
(Nanjing, China). RIPA Lysis Buffer and Enhanced BCA Protein Assay Kit were
purchased from Beyotime (Shanghai, China). Collagenase type II was purchased
from Worthington Biochemical Corp. (Lakewood, NJ, USA). Williams’ medium E was
obtained from Sigma-Aldrich Japan (Tokyo, Japan). Anti- Pro-caspase-3、Anti-
PRAP-1、Anti-β-actin receptor rabbit monoclonal antibody and rabbit IgG
monoclonal isotype control were purchased from Abcam (Cambridge, MA, USA). All
other chemicals made in China were analytical grade.Figure 1.

Chemical structure of Xanthohumol.

After anesthesia, mice were immediately perfused transcardially with 4% paraformaldehyde (PFA). The brains were fixed in 4% PFA for 24 h after perfusion, and then soaked in 15%, 25% and 30% sucrose for 24 h, 24 h and 48 h. Next, they were immerged in OCT compound and frozen at -80°C. After rinsing with PBST for three times, sections were incubated in blocking solution containing 0.2% Triton X and 5% goat serum for 40 min. Then they were incubated with primary antibody for the detection of PSD95 (1:200, Abcam), Synapsin-1 (1:200, Abcam), and p-Synapsin (1:200, Arigo) at 4°C overnight. Rabbit IgG monoclonal Isotype Control (1:200, Abcam) served as negative control. On the second day, sections were incubated in Goat Anti- Rabbit IgG (H&L) Cy3 secondary antibody (1:500; Abcam) for 2 h, followed by incubation with DAPI (1 μg/mL; Boster Bio, Wuhan, China) for 5 min. Images were captured using a fluorescence microscope (BX51, Olympus, Japan) and the immunopositive cells was analyzed by ImageJ software (version 1.52, NIH, USA) to calculate the calibrated total fluorescence with this formula: Integrated density - (Area × Mean background fluorescence), and then normalized to achieve fold changes (% of control).