Cy3 labeled goat anti mouse igg h l a0521

After the treatment, cells in the 24-well plate were fixed with 4% paraformaldehyde for 10–15 minutes at room temperature on a shaker (slow rotation). Triton X-100, 0.1%, was loaded into each well to allow antibodies to enter into cells. To reduce nonspecific binding, blocking solution was added for 2 hours at room temperature. Then, primary antibodies were added and incubated at 4°C overnight. Rabbit anti-NeuN (ab104225, 1:500) and mouse anti-β-III-tubulin (ab78078, 1:500) (both from Abcam, Shanghai, China) were used to label neurons and axons. After washing with PBS, secondary antibodies were added and incubated at room temperature in a dark box for 1 hour. Cy3-labeled goat anti-mouse IgG (H+L) (A0521, 1:500) and Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) (A0423, 1:500) were purchased from Beyotime (Shanghai, China). Cells were incubated with DAPI (nuclear dye) and then washed with PBS. Images were captured on a fluorescence microscope (TH4-200; Olympus). The axon length for each neuron was calculated using Image-Pro Plus software. NeuN-positive cells were counted in three random fields under the microscope.

The sections were routinely deparaffinized and hydrated, and then the sections were heated to 95°C in Tris-EDTA buffer (pH 9.0) for 15 min for antigen retrieval. Subsequently, the sections were incubated with 1% bovine serum albumin (BSA) in phosphate- buffered saline (PBS) for 30 min at 37°C to block nonspecific sites. Third, the sections were incubated respectively with different primary antibodies (data on the primary antibodies are described in Table 1) at 4°C overnight, followed by incubation with Cy3-labeled goat anti-rabbit IgG (H + L) (A0516, Beyotime, Jiangsu, China) for rabbit-derived primary antibodies, Cy3-labeled goat anti-mouse IgG (H+L) (A0521, Beyotime) for mouse-derived primary antibodies, or Cy3-labeled donkey anti-goat IgG (H+L) (A0502, Beyotime) for goat-derived primary antibody, at room temperature in the dark at 1:500 dilution for 1 h. Finally, the sections were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI; Beyotime) for 2 min at room temperature in the dark and mounted with anti-fade mounting medium (P0128, Beyotime). PBS was used for rinsing between steps. Sections omitting the primary antibodies or using normal serum of the same species as the primary antibodies were used as negative controls.