RPB1 immunofluorescence in HEK293 ARMC5 knockout cells

HEK293 cells or ARMC5 K.O. cells were fixed in 4% paraformaldehyde (EMS Emgrid 15710) for 15 minutes, then permeabilised in 0.25% Triton X100 (Sigma Aldrich 93443) for 10 minutes. Cells were incubated in 50% blocking buffer (Millenium Biosciences Li-Cor Intercept in PBS, LCR-927-70001) in PBS for 30 minutes, before being stained with primary antibodies in 50% blocking buffer in PBS for 90 minutes. Cells were then incubated for 30 minutes with secondary antibodies plus DAPI at 200 ng/mL in 50% blocking buffer in PBS. imaging was performed on a Nikon Ti2 microscope equipped with a Yokogawa CSU-W1 spinning disk, with 40x/NA0.95 Plan Apo λ air objective, and dual Hamamatsu ORCA-Fusion C14440-20UP cameras. 20 z-planes at 1 µm intervals were acquired. DAPI DNA stain was acquired with a 405 nm laser and 450/82 nm filter. Alexa488+ conjugated secondary antibodies were acquired with a 488 nm laser and 525/50 nm filter, and mCherry-RPB1 was acquired with a 561 nm laser and 617/73 nm filter. Where applicable, Alexa488-NHS or Alexa647-NHS cell stains were acquired with the appropriate green (525/50 nm) or far-red (685/40 nm) filter.将HEK293细胞或ARMC5基因敲除（K.O.）细胞用4%多聚甲醛（paraformaldehyde，货号EMS Emgrid 15710）固定15分钟，随后以0.25% Triton X-100（曲拉通X-100，Sigma Aldrich 93443）透化处理10分钟。将细胞置于50%体积比的封闭缓冲液（Millenium Biosciences Li-Cor Intercept溶于PBS，货号LCR-927-70001）与PBS的混合溶液中孵育30分钟，之后在相同比例的封闭缓冲液-PBS混合体系中与一抗共同孵育90分钟。随后将细胞与含终浓度200 ng/mL DAPI的二抗工作液（以同样的50%封闭缓冲液-PBS混合体系配制）共同孵育30分钟。成像实验采用配备Yokogawa CSU-W1转盘的Nikon Ti2显微镜完成，搭配40×/NA0.95 Plan Apo λ空气物镜及两台Hamamatsu ORCA-Fusion C14440-20UP相机。采集20层Z轴平面，层间距为1 µm。DAPI DNA染色通过405 nm激光与450/82 nm滤光片采集；Alexa488标记的二抗通过488 nm激光与525/50 nm滤光片采集；mCherry-RPB1通过561 nm激光与617/73 nm滤光片采集。若需检测Alexa488-NHS或Alexa647-NHS细胞染色，则分别使用适配的绿色通道（525/50 nm）或远红外通道（685/40 nm）滤光片完成采集。