Duolink in situ detection reagent red

Duolink in situ detection reagent red (DUO92008; Sigma) was used to perform PLA in accordance with the manufacturer’s protocol. Briefly, cells were washed twice with 1X PBS, cytospun on a glass slide, fixed, permeabilized, and then incubated with blocking buffer (all reagents provided in the kit) for 1 hour. Subsequently, the cells were probed with primary antibodies against MLL (14197, Cell Signaling Technology, 1:250), UBTF (F-9, Santa Cruz #SC-13125; 1:50), and menin (AbCam ab2605, 1:250). For negative reaction controls, cells were incubated with only 1 of each antibody. The slides were washed and incubated (1 hour, 37°C), with specific plus and minus Duolink PLA probes (1:5). After washing, the slides were further incubated with ligation-ligase solution (30 min, 37°C) followed by incubation with amplification polymerase solution (2 h, 37°C). The slides were finally incubated with DAPI (300 nM) for 5 minutes in the dark and washed twice. Images were acquired using a confocal microscope (Leica, SP8) and processed by Leica LAS AF Lite software (Leica). For flow cytometry analysis of PLA, all the steps were performed in 96-well plates, and data were acquired by a Beckman Coulter CytoFLEX LX with subsequent data analysis using FlowJo software (V10.0.7; TreeStar, Ashland, OR).