Donkey anti rabbit fluorescent secondary antibody

HT22 cells were fixed with 4% paraformaldehyde (PFA) for 15 min, blocked with 10% donkey serum for 1 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: rabbit anti-PSD95 (cat#: ab18258, Abcam, USA), rabbit anti-phospho-ERK1/2 (cat#: 9101, cell signaling, USA), and rabbit anti-phospho-eIF4E (cat#: ab76256, Abcam, USA). The next day, the cells were incubated with donkey anti-rabbit fluorescent secondary antibody (cat#: A21207, Invitrogen, USA) for 2 h at room temperature in the dark. They were then counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (cat#: C0065, Solarbio, China) for 10 min and sealed with anti-fluorescence quenching sealing tablets (cat#: S2100, Solarbio, China). Images were taken using a laser confocal microscope (Olympus, Japan), and the average optical density was analyzed using Fiji software (National Institutes of Health, USA). Intra and inter-assay coefficients of variation were 2.44%-4.95% and 4.07%-9.81% respectively.

HT22 cells were fixed with 4% paraformaldehyde at room temperature for 15 min, blocked in 10% donkey serum at room temperature for 1 h, and incubated overnight at 4°C with the following primary antibodies: anti-PSD95 (cat#: ab18258, Abcam, United States), anti-FMRP (cat#: ab17722, Abcam, United States). Afterward, the cells were incubated with donkey anti-rabbit fluorescent secondary antibody (cat#: A21207, Invitrogen, United States) at room temperature in the dark for 2 h, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (cat#: C0065, Solarbio, China) for 10 min, and sealed with anti-fluorescence quenching sealing tablets (cat#: S2100, Solarbio, China). Images were photographed using a laser confocal microscope (Olympus, Japan), and the average optical density was analyzed using the Image J software (National Institutes of Health, United States).