Alexafluor488 azide

EdU was given by intraperitoneal injection at a concentration of 5 µg g⁻¹. For EdU staining, sections were permeabilized by incubation for 30 min in 0.5% Triton X-100 in PBS followed by 3 × 5-min washes in PBS. EdU staining solution was made fresh each time, containing 100 mM Tris, 4 mM CuSO₄, and 100 mM ascorbic acid diluted in 1× PBS. The fluorescently labeled azide molecule was added last, and the staining cocktail was immediately added to sections for a 30-min incubation at room temperature (final concentrations of each fluorescent molecule: 4 mM AlexaFluor488-Azide [Click Chemistry Tools, 1275-1], 10 mM Cy3-Azide [Lumiprobe, A1330], 16 mM AlexaFluor647-Azide [Click Chemistry Tools, 1299-1]). After incubation, sections were washed 3 × 5 min in PBS. After EdU labeling, standard protocols were followed for immunostaining.

Cells were seeded at 100,000/well in 24-well plates the day before, then incubated with 500 μM 5-EU (Click Chemistry Tools No. 1261) diluted in complete media for 2 h, rinsed, lifted with Accumax, and fixed in 1% PFA. Following fixation, the cells were permeabilized by PBS supplemented with 3% bovine serum albumin (BSA) and 0.1% saponin for 15 min. 5-EU was labeled in permeabilized cells using the Click & Go kit (Click Chemistry Tools No. 1263) with 2 μM Alexa Fluor 488 azide (Click Chemistry tools No. 1275) according to the manufacturer’s instructions. Labeled cells were washed twice and resuspended with PBS containing 3% FBS prior to analysis by flow cytometry.