LAMP-1 Antibody (H4A3)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Dilute at 1 : 80 diluted in PBS. - Incubate cells at 4 °C overnight,

Protocol tips
- Dilute at 1 : 80 diluted in PBS. - Incubate cells at 4 °C overnight,

Publication protocol

Cells were seeded on sterile coverslips placed in 24-well plates. After incubating with 2.5 μM Cd and/or 5 mM Tre for 12 h, cells were fixed with 4% paraformaldehyde for 8 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min and blocked with 2% bovine serum albumin in PBS for 1 h at room temperature. Slides were first stained with anti-LC3 antibody (1 : 150 diluted in PBS) at 4 °C overnight. After washing the cells with PBS, cells were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1 : 600 diluted in PBS) for 1 h at room temperature and washed with PBS again. Subsequently, cells were stained with anti-LAMP-1 antibody (1 : 80 diluted in PBS) at 4 °C overnight, washed with PBS again and incubated with Alexa Fluor 555-conjugated goat anti-mouse secondary antibody (1 : 500 diluted in PBS) for 1 h at room temperature. Nuclei were stained with DAPI (blue). Finally, all slides were mounted with ProLong Gold Antifade Mountant. Images were conducted on the Leica TCS SPE confocal microscope with a × 63 (1.3 numerical aperture) oil-immersion objective. Images for colocalization analysis (percentage of protein–protein colocalization) were assessed using the JaCoP plugin in ImageJ after thresholding of individual frames.64 All colocalization calculations were performed on three independent experiments with 50 cells per condition in each experiment. Images were prepared for presentation using Adobe Photoshop 6.0 (San Jose, CA, USA).

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Manufacturer protocol

Download the product protocol from Santa Cruz Biotechnology for LAMP-1 Antibody (H4A3) below.

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