Tritc conjugated anti rabbit igg

Immunofluorescence was used to evaluate the distribution and expression of LC3-II, Beclin1, and P62 in SH-SY5Y cells from normal control and OGD/R groups. For this purpose, the cells were incubated with antibodies against LC3 (1:800; Novus; NB600-1384), Beclin1 (1:100; Abcam Cat# ab55878) and P62 (1:100; Abcam Cat# ab91526), respectively, in a humidified container at 4°C for 12 hr. The cells were rinsed in PBS for 3 times, and incubated with TRITC conjugated anti-rabbit IgG (1:100, Proteintech) at room temperature for 4 hr. 4, 6-diamidino-2-phenylindole (DAPI, 0.0001%, Sigma) was applied to stain nuclei. The cells were examined by a laser confocal microscope (Nikon D-Eclipse C1, Japan).

Cells were fixed in cold methanol at −20°C. After blocking in 5% normal goat serum, cells were incubated with the primary antibody against LC3B (#3868; 1:100, Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight and then TRITC-conjugated anti-rabbit IgG (#SA00007-2; 1:100, Proteintech, Chicago, IL, USA) for 2 h at room temperature (Li et al., 2017 (link)). Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, # D9542; Sigma Aldrich, St. Louis, MO, USA). Images of cells were gained via a laser-scanning confocal microscope (Nikon, Japan).

Cells were fixed with cold methanol at −20°C for 15 min and washed three times in phosphate‐buffered saline (PBS) for 5 min each. Cells were blocked in 5% normal goat serum for 1 hr at room temperature. The primary antibody against LC3B (1:100, Cell Signaling Technology, Beverly, MA, USA) was incubated at 4°C overnight. Cells were incubated with 1:100 TRITC‐conjugated anti‐rabbit IgG (Proteintech, Chicago, IL, USA) at room temperature for 2 hr. Nuclei were viewed with 4',6‐diamidino‐2‐phenylindole (DAPI) (Sigma‐Aldrich, St. Louis, MO, USA) after 5 min at room temperature. Cells were imaged using a laser scanning confocal microscope (Nikon, Japan).