Alexa fluor 488 conjugated f ab 2 fragment of goat anti mouse igg

The cells were lysed with 1% NP-40 in a solution of 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.01 M MgCl₂. The cell debris was removed by centrifugation at 16,000 ×g for 20 minutes. The supernatants were removed, and the protein content was quantified using a BCA assay. An aliquot of 20 µg of protein was loaded into each well of an SDS-PAGE gel. For immunoblotting, the SDS-PAGE gel was electroblotted onto a PVDF membrane. An anti-TYRP-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH antibody (Santa Cruz Biotechnology), anti-MART-1 antibody (Thermo Fisher Scientific, CA, USA), anti-HMB45 antibody (GeneTex, Irvine, CA, USA), anti-TA99 antibody (GeneTex), anti-PMEL17 (aN) (GeneTex) and anti-TYR antibody (Upstate Biotechnology, Lake Placid, NY, USA) were used for protein detection. For the fluorescence microscopy, cells were cultured on Lab-Tek chamber slides (Nunc, NY, USA), fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 5 minutes, and incubated with a primary antibody. Fluorescence was detected by secondary antibody staining with the Alexa Fluor 488-conjugated F(ab')2 fragment of goat anti-mouse IgG (Invitrogen) and the Alexa Fluor 594-conjugated F(ab')2 fragment of goat anti-rabbit IgG (Invitrogen).

Cells were cultured on Lab-Tek chamber slides (Thermo Fisher Scientific), fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), and permeabilized with 0.1% Triton X-100 in PBS containing 1% BSA for 5 min. Afterwards, the cells were incubated with anti-PMEL (Invitrogen, Waltham, MA, USA), anti-LCB antibody (Cell Signaling Technology), and DAPI (Molecular Probes, Eugene, OR, USA). Fluorescence was detected by secondary antibody staining with Alexa Fluor 488-conjugated F(ab’)2 fragment of goat anti-mouse IgG and Alexa Fluor 594-conjugated F(ab’)2 fragment of goat anti-rabbit IgG (Invitrogen). All images were acquired using an LSM 800 confocal microscope (Zeiss, Jena, Germany). The fluorescence intensity of PMEL and LC3 co-expression was quantified using ImageJ, and displayed in corrected total cell fluorescence (CTCF). Graphs depict the quantification of cell integrated density among CTL, 2'-FL, DMP, and 2'-FL + DMP in the four experimental groups. To measure intensity, total cell fluorescence (CTCF) was calculated using the formula: CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings).