Cathepsin b pa5 17007

Cells were grown on coverslips, fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X100 for 10 min, washed three times with PBS and incubated with primary antibody overnight at 4 °C. The cells were washed three times with PBS and incubated with secondary antibody for 1 h at room temperature. After washing, coverslips were mounted using Prolong Gold antifade reagent with DAPI (Invitrogen). Paraffin-embedded tissue sections were cleared with histoclear (National Diagnostics) and graded alcohol using standard techniques. Antigen retrieval was performed using 7 mM citrate buffer, pH 6.0 under pressure. Sections were incubated with primary antibody overnight at 4 °C and with secondary antibody for 1 h at room temperature. Coverslips were mounted using Prolong Gold antifade reagent with DAPI (Invitrogen). All images were obtained using a Zeiss 780 confocal microscope and Zeiss LSM 880, and settings were adjusted to allow for detection of fine membrane structure. Primary antibodies were against: LAMP2 (ab13524) from Abcam (Cambridge, MA); PMCA2 (PA1-915) from Thermo Scientific (Waltham, MA); cathepsin B (PA5-17007) and TFEB (PA5-96632) from Invitrogen (Grand Island, NY); Phospho-STAT3 (9145), p21 (2947), p-Rb (8516), and NFAT (5861) from cell signaling (Danvers, MA); PCNA (sc-25280) from Santa Cruz (Dallas, TX).

Cells were grown on coverslips, fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X100 for 10 mins, washed 3 times with PBS and incubated with primary antibody overnight at 4°C. The cells were washed 3 times with PBS and incubated with secondary antibody for 1 hour at room temperature. After washing, coverslips were mounted using Prolong Gold antifade reagent with DAPI (Invitrogen). Paraffin-embedded tissue sections were cleared with histoclear (National Diagnostics) and graded alcohol using standard techniques. Antigen retrieval was performed using 7mM citrate buffer, pH 6.0 under pressure. Sections were incubated with primary antibody overnight at 4°C and with secondary antibody for 1 hour at room temperature. Coverslips were mounted using Prolong Gold antifade reagent with DAPI (Invitrogen). All images were obtained using a Zeiss 780 confocal microscope and Zeiss LSM 880, and settings were adjusted to allow for detection of fine membrane structure. Primary antibodies were against: LAMP2 (ab13524) from Abcam (Cambridge, MA); PMCA2 (PA1-915) from Thermo Scientific (Waltham, MA); cathepsin B (PA5-17007) and TFEB (PA5-96632) from Invitrogen (Grand Island, NY); Phospho-STAT3 (9145), p21 (2947), p-Rb (8516) and NFAT (5861) from cell signaling (Danvers, MA).