Alexa fluor 488 goat anti rabbit igg h l

SH-SY5Y cells were grown on poly-l-lysine coated coverslips to improve cell attachment in 6-well culture plates at a density of 1.5 × 10⁵ cells per well. Then, cells were pretreated with Andro at designated concentrations for 24 h, followed by exposure to 1.5 mM MPP⁺ for 16 h. After that, treated cells were incubated with MitoTracker Red (Cell Signaling) for 30 min at 37 °C, then fixed with cold methanol for 15 min at −20 °C and washed three times with PBS for 5 min. Treated cells were permeabilized with 0.25% Triton-X-100 in PBS and blocked with 1% bovine serum albumin, 10% normal goat serum, and 0.3% glycine in PBST for 2 h. Subsequently, cells were incubated with anti-LC3 primary antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:200 in a dark humidity box at 4 °C overnight, then incubated with a secondary antibody (Invitrogen, Alexa Fluor 488) goat anti-rabbit IgG (H+L) at 1:500 dilution for 2 h. Finally, the images were taken with a fluorescence microscope (Olympus BX53, Tokyo, Japan).

Five mice from each group were anesthetized and perfused with an intracardial infusion of saline, followed by 4% paraformaldehyde. Brains were subsequently post-fixed in the same fixative for 48 h and then dehydrated in 20% and 30% sucrose, successively, at 4 °C. Brains were sectioned at a thickness of 16 μm on a sliding microtome for immunofluorescence (Leica CM1520, Solms, Germany). Sections were incubated with 0.2% Triton X-100 for 15 min at 25 ± 5 °C and blocked in 5% goat serum for 30 min at 37 °C. Then, the sections were stained with a primary antibody for against tyrosine hydroxylase (TH) (1:100) overnight at 4 °C. After washing three times with PBS, sections were incubated with Alexa Fluor ^®® 488 goat anti-rabbit IgG (H+L; Eugene, OR, USA; 1:200) for 1 h. Positive neurons were detected in five to six sections using a scanning confocal microscope (Olympus Fluoview FV1000). Fluorescence intensity was quantified using Image J software. The average fluorescence intensity was calculated from 6–8 distributed areas in the sample images.