Study design

LC3B  stainingin human Monocytes 

Aim : To test the expression of LC3B in human Monocytes.

Buffer :

10x PBS 

BSA free fatty acid

LC3B rabbit ployclonal antibody (Novus, NB100-2220, store it at -20°C)

Triton X 100 (Sigma, X-100, RT)

Image IT fix signal engancer (Invitrogen, Ref I36933, 4°C).

Prolong Gold + DAPI (Invitrogen, Ref P36935, -20°C).

Goat anti rabbit IgG Alexa fluor 488 (Invitrogen, A11008, store at 4°C).

Antibody Diluent solution, (Dako,  REF S2022, store at 4°C).

Protocol :

Wash the cells by cold 1xPBS x3.

Fix the cells by 4% PFA for 10min  at RT.

Wash the cells by 1xPBS for 5min x3.

Block the cells in normal 1%BSA for 1h at RT (20ul Triton X100 + 10ml PBS, mix well).

Incubate the sections in primary antibody LC3B (1 :200, 5μl into 1ml Antibody Diluent solution)  for 2 h at RT.

Wash the cells by 1xPBS for 5 mins x3.

Incubate the cells in Image IT fix, 4 drops each well for 30min at RT.

Incubate the cells with secondary antibody goat anti-rabbit 488 (1 :1000, 1μl into 1ml Antibody Diluent solution) for 30 min at room temperature (avoid the light, in the dark room). 

Wash the cells by 1x PBS for 5min x3 (avoid the light, in the dark room).

Add an antifade mounting medium onto the cells ( ProLong Gold + DAPI).

Wait for 24hr in the flat and dark place at RT, then take the photos by confocal microscopy (Zeiss).

Seal the coverslip by nail polish.

Store at –20°C in the slide box.