Alexa fluor 488 donkey anti rabbit igg secondary antibody

NPC cells were fixed with 4% paraformaldehyde and permeabilized by 0.2% Triton X-100. After 1 h blocking with 1% bovine serum albumin at room temperature, the cells were incubated with anti-STAT1 (1:100) antibody overnight at 4 °C. Next day, the cells were washed off the unbound primary antibody and stained with donkey anti-rabbit IgG Alexa Fluor 488 secondary antibody (ThermoFisher, #A32790) for 1 h at room temperature.

Cells were treated with DMSO (1% v/v), TGFβ1 (5 ng·mL⁻¹), and/or NOX1/4 inhibitor (GKT137831) (20 µm) during 24h in N2B27 medium. After treatment, cells were washed with PBS and fixed with 3.7% (w/v) paraformaldehyde in PBS for 15 min. Subsequently, cells were washed with PBS and blocked with 10% FBS in PBS with 1% BSA for 1 h, at RT. Then, cells were permeabilised with 0.1% Triton‐X‐100 in 0.1% BSA for 5 min. Next, cells were incubated at RT for 2 h with primary antibodies against Ki67 (1 : 1000 in PBS containing 1% BSA, rabbit, ab15580; Abcam^®). Afterwards, cells were washed in PBS and incubated for 1 h in dark at RT with donkey anti‐Rabbit IgG Alexa Fluor 488 secondary antibody (1 : 200 in PBS with 1% BSA, A21206; Thermo Fisher Scientific). Then, cells were incubated in dark at RT for 5 min with DAPI (1 : 1000 in PBS with 1% BSA; Sigma‐Aldrich Co.). Later, cells were washed three times with PBS and the coverslips were mounted on the slides using Fluoromont‐G^® mounting medium (Southern Biotech, Birmingham, AL, USA) and dried in dark at RT. Pictures were acquired with the fluorescence microscope Eclipse 90i and processed using NIS‐Elements software (Nikon, Tokyo, Japan). The quantification of the proliferative phenotype of cells was performed by quantifying Ki67‐positive cells, and total number of nuclei per picture using fiji image j software [35 (link)].

SH-SY5Y cells were plated on glass coverslips, previously coated with poly-l-lysine (Sigma-Aldrich), for 30 min at RT, and incubated for 24 h in DMEM media before the drug treatments. The cells were fixed with 4% paraformaldehyde/DPBS for 20 min at RT, washed three times with DPBS, and blocked in DPBS containing 5% (v/v) FBS and 0.3% (v/v) TX-100 for 1 h at RT. After further washings, the cells were incubated for 1 h in the antibody solution (D-PBS, 1% (w/v) BSA, 0.3% (v/v) Tx-100) with the rabbit anti-TFEB primary antibody (1:600, Bethyl Laboratories, Cat. n. A303-673A). After being washed, the cells were incubated with the donkey anti-rabbit IgG Alexa Fluor^®488 secondary antibody (Thermo-Fisher, Cat. n. A-21206) for 1 h in an antibody solution. Successively, the coverslips were mounted on glass slides using Vectashield with DAPI (Vector Laboratories Inc. Burlingame, CA, USA), and fluorescence microscopy analysis was performed using a Nikon TE2000 microscope (Nikon Instruments S.p.A, Florence, Italy). Image processing was performed by using Adobe Photoshop CS software (Adobe Systems Incorporated).