Goat polyclonal anti mouse igg alexa488 conjugate

Cells were cultured on cover slips in a 12-well plate, fixed with ice-cold 1:1 acetone: Methanol solution for 30 s followed by 5-s washing with PBS, and blocked with 1% (w/v) BSA/1% (w/v) gelatin in PBS overnight in 4°C. Primary antibodies used in immunoblotting were mouse monoclonal anti-rabbit anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse monoclonal anti-SSAO 7–106 (AK 982/02), mouse anti-Syn6 (BD Transduction Laboratories, 610636), and goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001). Secondary antibodies were goat polyclonal anti-rabbit IgG Alexa488-conjugate (Thermo, A11034) and goat polyclonal anti-mouse IgG Alexa488-conjugate (Thermo, A31570). All antibodies were used at a dilution of 1:100. Cells were imaged with a Zeiss LSM 710. Images were processed with ZEN and Image J.

Immunostaining was performed as previously described9 (link). Cells were cultured on collagen-coated cover slips in a 12-well plate, fixed with 3.7% paraformaldehyde in PBS for 20 min, permealized with 0.2% (w/v) Triton X-100 in PBS for 10 min, and blocked with 1% (w/v) BSA/1% (w/v) gelatin in PBS. Primary antibodies used in cell staining were mouse monoclonal anti-FLAG M2 clone (Sigma, F1804), rabbit polyclonal anti-ATP7A (Hycult Biotech, HP8040), mouse monoclonal anti-ATP7A (Santa Cruz, 376467), mouse monoclonal anti-EEA1 (BD Biosciences, 610456), mouse monoclonal anti-Rab11 (BD Biosciences, 610656), mouse monoclonal anti-Lamp1 (developmental studies hybridoma bank, H4A3-s), and sheep monoclonal anti-TGN46 (Gene Tex, GTX74290). Secondary antibodies used for cell staining were: goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001), goat polyclonal anti-rabbit IgG Alexa555-conjugate (Molecular Probes, A-21428). Cells were imaged with a Zeiss LSM 710. Images were processed with ZEN and Image J. Anti-EEA1, anti-Rab11, and anti-Lamp1 antibodies were kindly provided by Dr Rajini Rao (Johns Hopkins University). All antibodies were used at a dilution of 1:200.