BiP-3xFLAG HEK293 cells (0.12 × 106) were plated on 12-mm round glass coverslips (Thermo Fisher Scientific) coated with 0.15 mg/ml poly-lysine in 24-well plates. The following day, cells were fixed and immunostained using a standard procedure (Sundaram et al., 2017 (link)). Rat anti-FLAG antibody (#637303; Biolegend) and mouse α-PDI (#MA3-018; Affinity Bioreagents) were used as primary antibodies. Goat α-RAT IgG-Cy2 (#112-225-167; Jackson ImmunoResearch) and Goat α-Mouse IgG-Alexa657 (#A-21235; Invitrogen) were used as secondary antibodies. Cells were imaged on Leica scanning confocal consisting of an inverted microscope (Leica SP6/SP8) and an HC PL APO 63×/1.4 (CS2 No: 11506350) oil objective lens (Leica) and was controlled by the Leica Application Suite X software. Sequential image scanning at 1× zoom, 100 Hz, 1,024 × 1,024 pixels, and with line averaging set at four was used to collect images for displayed images.
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Visualizing BiP-3xFLAG in HEK293 Cells
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Corresponding Organization : Yale University
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Variable analysis
independent variables
- Plating density of BiP-3xFLAG HEK293 cells (0.12 × 10^6 cells)
- Immunostaining of BiP-3xFLAG HEK293 cells using anti-FLAG and anti-PDI antibodies
- 12-mm round glass coverslips coated with 0.15 mg/ml poly-lysine
- Standard immunostaining procedure
- Leica scanning confocal microscope settings (1× zoom, 100 Hz, 1,024 × 1,024 pixels, line averaging set at four)
- Positive control: Not specified
- Negative control: Not specified
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