Cfi apo lwd objective

Click-iT^® Plus TUNEL Alexa^® 594 In Situ Apoptosis Detection Kit (Invitrogen) was used. Twenty-four hours after bortezomib treatment, cells were rinsed once with PBS, fixed with 4% PFA, and processed following the 3D immunofluorescence staining protocol. After TritonX-100, glycine and IF buffer washes, cells were washed twice in deionized water and incubated with the TdT Reaction buffer for 10 minutes at room temperature. Samples were then incubated with the TdT Reaction mixture overnight in a humidified chamber at room temperature. Cells were washed in IF buffer three times for 20 minutes each, once with PBS and processed with the Click-iT Plus TUNEL reaction cocktails for 30 minutes at room temperature in the dark. Samples were counterstained with DAPI and mounted with Prolong Diamond Antifade reagent. Samples were analyzed using a Leica DMI 3000 fluorescent microscope with a 20X objective. Images were acquired using a Nikon A1RMP confocal microscope with a 25X CFI Apo LWD Objective. Statistical significance was determined by two-way ANOVA.

Click-iT^® Plus OPP Alexa Fluor^® 594 Protein Synthesis Assay Kit (Invitrogen) was used. Forty-eight hours after doxycycline induction, media were replaced with fresh media containing 20 μM Click-iT OPP (O-propargyl-puromycin) reagent and incubated in the cell culture incubator for 30 minutes. Cells were rinsed once with PBS, fixed with 4% PFA, and processed following the immunofluorescence staining protocol above. After TritonX-100, glycine and IF buffer washes, Click-iT^® reaction was performed according to the vendor protocols. Samples were counterstained with 1X HCS NuclearMask Blue Stain and mounted with Prolong Diamond Antifade reagent. Images were acquired using a Nikon A1RMP confocal microscope with a 25X CFI Apo LWD Objective.