The Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit

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	Membranes were blocked with 5% skim milk in TBS-Tween (TBST) for 1hr and then incubate with primary antibodies diluted in 1% (w/v) skim milk in TBST for 2hrs shaking at RT or overnight shaking at 4°C

Protocol tips
Membranes were blocked with 5% skim milk in TBS-Tween (TBST) for 1hr and then incubate with primary antibodies diluted in 1% (w/v) skim milk in TBST for 2hrs shaking at RT or overnight shaking at 4°C

Publication protocol

Confocal microscopy
MLO-Y4 cells cultured on glass coverslips and calvarial primary osteocytes ex vivo cultured in 96-wells plates were fixed for 15mins and and 2hrs at RT with 4% PFA respectively. Cells were then permeabilized for 5 minutes with 0.1% Triton X-100 in PBS, and non-specific antibody binding was blocked by 3% BSA-PBS for 30mins. MLO-Y4 cells and primary calvarial osteocytes were incubated with primary antibody diluted in 0.2% BSA-PBS for 2hrs at RT or overnight at 4°C respectively. Following incubation cells were washed extensively and bound primary antibodies were reacted with fluorescence conjugated secondary antibody for 1hr at RT, and nuclei were stained with Hoechst 33258 dye for 15mins at RT. Coverslips were then mounted in ProLong Diamond Anti-fade medium (Invitrogen, USA). Images were acquired on a Nikon A1 confocal microscope equipped with 60X (oil immersion) lens, and analyzed using ImageJ software. For quantification of the number and length of dendritic processes and the number of Cx43 fluorescence dots and endogenous LC3GFP punctual of each osteocyte, the channels were extracted and converted to greyscale images and analyzed using ImageJ software.

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