Plan apochromat 100x 1.46 oil objective

Thin smears made from the synchronous culture of Pfcyp19b-OE lines were fixed using paraformaldehyde (Sigma) (4%) and glutaraldehyde (Sigma) (0.0075%) in 1xPBS (1^st Base) for 15 min, permeabilized in Triton X-100 (Sigma) (0.1%) for 10 min and then blocked with bovine serum albumin (BSA, Sigma) (3%) with glycine supplementation (1st Base) (22.52mg/ml) in PBS-T for 1 hour. Subsequently they were incubated with 1:200 rat monoclonal anti-HA antibody (Roche) and 1:200 rabbit polyclonal anti-PfBiP (BEI Resources) followed by incubation for 30 minutes with 1:2000 Alexa Fluor 594 goat highly cross-absorbed anti-rat (Jackson Immunoresearch) and 1:2000 Alexa Fluor 488 goat highly cross-absorbed anti-rabbit secondary antibodies (Jackson Immunoresearch). Nuclear counterstain was done with DAPI (Thermo Scientific) (1 ng/μl) for 10 min. All steps were performed at room temperature. Slides were mounted with ProLong Gold Antifade (Thermo Scientific). Images were captured with Zeiss LSM710 confocal microscope equipped with an Airyscan detector using a Plan-Apochromat 100x/1.46 oil objective and processed using Zen (Blue edition) imaging software (Zeiss). Images were analyzed using ImageJ. All antibodies have been tested for cross-reactivity and corresponding empty Vector control strain at the same generation and developmental stage was used parallelly as a negative control.

SIM was performed on Elyra PS1 microscope (Carl Zeiss), with Plan Apochromat 100x/1.46 Oil objective, 1.6x lens in the detection light path and Andor iXon 885 EMCCD camera; image pixel size was 50 nm; SIM raw images were acquired with 3 grid rotations and 5 phases, at 50 ms exposure for each orientation and phase combination. In multi-color images, channels were acquired sequentially using standard single-band filter sets. Z-stacks were acquired with 100 nm intervals. Raw SIM images were processed with Zen Black 2012 software (Carl Zeiss). 2D time lapse series of 50–100 frames were acquired at the speed of 1.8 s per frame, in SYBR Gold channel only. Confocal imaging for comparison with SIM was performed using the LSM light path of Elyra PS1 microscope, which enabled acquisition of the same fields of view in confocal and SIM modes, using Plan Apochromat 100x /1.46 Oil objective, with pixel size matched to that of SIM images.