Achroplan 40 75w objective

Raw264.7 cells were fixed with 3.7% methanol-free formaldehyde, and F-actin formation in Raw264.7 cells was observed after staining with Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific) according to manufacturer’s instructions. Images were captured with an epifluorescence Zeiss (White Plains, NY) Axioskop and a Zeiss (Achroplan 40/.75W) objective.

NSCLC cells were fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X‐100, and washed with PBS before incubation for 2 h in 5% BSA at 25°C to facilitate blocking. Samples were then probed overnight at 4°C with anti‐NFE2L1 (ab238154, Abcam, 1: 200) in BB, followed by washing with PBS and staining at room temperature for 1 h with AF488‐conjugated goat‐anti‐mouse IgG (1: 100). The epifluorescence Zeiss Axioskop and a Zeiss (Achroplan 40/.75 W objective) were used to capture uncompressed 24‐bit TIFF files. A cooled (−12°C) single CCD color digital camera (Pursuit, Diagnostic Instruments) driven by the freely available SPOT version 4.7 software was utilized for this purpose. To detect AF488 (Chroma Technology), a filter set 41,001 with high IQ was employed. To determine the ratios of nuclear/cytoplasmic, the measurement of average fluorescence intensity was conducted for NFE2L1 localization in both the nucleus and the immediate surrounding area (cytoplasm). Zeiss analysis imaging software, which is freely available, was utilized for conducting all Image analysis. Eighty percent of the population was represented by the chosen cells, which were shown in the relevant figures.