Primary antibody against bcl 2

Proteins were extracted from cells using RIPA lysis buffer (Beyotime, China), and the concentrations were measured using BCA protein Assay (Beyotime, China). Proteins were separated using 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). After blocked in 5% nonfat milk and incubated with primary antibody against Bcl-2, Bax, pro-Caspaes-3, cleaved-Caspase-3, NLRP3, and GAPDH (Abcam, USA) overnight at 4°C, the membranes were incubated with HRP-conjugated secondary antibody at the room temperature for 1 hour. The bands were detected with the ECL detection system (Millipore, USA) and exposure to X-ray film.

The tumor samples from CRC patients were fixed in 10% formaldehyde, embedded in paraffin and then sectioned into slices. For IHC assay, tumor slices were firstly deparaffinized and rehydrated. After washing, slices were treated with H₂O₂ to reduce the endogenous peroxidase activities. Slices were then incubated with primary antibody against BCL2 (1:50, Abcam, Cambridge, UK) overnight at 4  °C. With three times washing in PBS, the slides were incubated with secondary streptavidin–horseradish peroxidase-conjugated antibody (1:3000, Abcam, Cambridge, UK) for 1 h, and reacted with 3,3-diaminobenzidine tetrahydrochloride (DAB) solution (Yeasen Biotech, Shanghai, China) for 5 min. Finally, slides were counterstained with hematoxylin solution (Beyotime Biotechnology, Shanghai, CHN) for 1 min, dehydrated and mounted with neutral gum.

Poly-L-lysine (PLL) (10x) was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). DMEM/F12 (1 : 1), Neurobasal Plus Medium, B27 Plus Supplement (50x), HBSS (1x), FBS, and 0.25% trypsin were obtained from Gibco (Grand Island, New York, USA). The Aβ_25-35 fragment and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). Donepezil hydrochloride, SB203580, and U46619 were obtained from Santa Cruz Biotechnology Inc. (Delaware, USA). L-Glutamine was obtained from Thermo Fisher (Massachusetts, USA). The rabbit polyclonal antibodies against MAP2 were purchased from Proteintech Group, Inc. (Chicago, USA). Rabbit monoclonal antibodies against p38 MAPK, p-p38 MAPK, caspase-3, cleaved caspase-3, and Bax were purchased from Cell Signaling Technology (Beverly, MA, USA). The primary antibody against Bcl-2 was purchased from Abcam (Cambridge, UK). Horseradish peroxidase- (HRP-) conjugated IgG secondary antibody was purchased from Beijing Bioss Biotechnology Co., Ltd. (Beijing, China), and the BCA protein assay kit was obtained from Beyotime Biotechnological Technology Co., Ltd. (Shanghai, China).