Immunofluorescence Analysis of Lysosomal and Autophagic Markers in hDF Cells

hDF cells (20,000 cells in 24-well plates on coverslip) were grown in complete medium for 24 h. Starvation was performed for 1 h in Hanks’ Balanced Salt solution (HBSS) before fixing with paraformaldehyde (PFA) 4% and staining with anti-LAMP1, anti-LAMP2, anti-p62, anti-LC3B, anti-TMEM175, anti-GC, and anti-GM antibodies. Briefly, incubation with blocking buffer (1 × PBS/0.1% triton/5% serum) was performed for 15 min in a humidified chamber. Primary and then secondary antibody incubations (1 × PBS/0.1% triton/antibody) were performed at 4 °C overnight and at RT for 1 h, respectively. Anti-LAMP1 (ab25630, Abcam, Cambridge, UK, 1:20), anti-LAMP2 (ab25631, Abcam, Cambridge, UK, 1:100), anti-LC3B (2775, Cell Signaling, Danvers, MA, USA, 1:200), anti-TMEM175 (19,925–1-AP, Proteintech, Manchester, UK, 1:100) and anti-p62/SQSTM1 (P0067, Sigma-Aldrich, St. Louis, MO, USA, 1:500), anti-GlcCer (RAS0010, Glycobiotech, Kuekels, Germany, 1:50) primary antibodies and anti-mouse IgG-Cy3 (C2181, Sigma-Aldrich, St. Louis, MO, USA, 1:200), anti-rabbit IgG-Cy3 (C2306, Sigma-Aldrich, St. Louis, MO, USA, 1:200), Donkey anti-Mouse IgG (H + L), Alexa Fluor™ 488 (A-21202, Thermo Fisher Scientific CA USA, 1:400), and Donkey anti-Rabbit IgG (H + L) Alexa Fluor™ 488 (A-21206, Thermo Fisher Scientific CA USA, 1:400) secondary antibodies were used. GM1 was detected through 2-h incubation with Cholera toxin subunit B (CT-B) conjugated with Alexa fluor 488 (c34775, Molecular Probes, Eugene, OR, USA, 1:50). Hoechst H3570 (Thermo Fisher Scientific, CA, USA, 1:1000) was used to visualize the nuclei. Slides were visualized with Nikon Confocal Microscope A1R and with Nikon Eclipse Ni-E Microscope.