Elyra s1 microscope

For images shown on Figure 1C, immunostaining was performed as described above, but slides were mounted using ProLong Diamond mounting media and covered with Zeiss high-performance 0.17 ± 0.005 coverslips. Images were acquired using a Zeiss Elyra S1 microscope, processed with Fiji, and mounted in Photoshop.

SIM was performed on a Zeiss Elyra S.1 microscope with a 63× oil-immersion objective (NA 1.40). Z-stacks of 15 to 20 equidistant planes were acquired. For each field of view, nine images were acquired using three different rotations and phases of structured illumination (a grid pattern) on the sample.

HCT-8 cells were seeded onto six-well plates containing high-precision cover glasses, and grown to 60 to 70% confluence. Bleached and washed transgenic oocysts were used for infecting the HCT-8 monolayer for 24, 48, or 72 h. Cells were fixed with 4% paraformaldehyde in PBS, followed by permeabilization with 0.25% Triton X-100 in PBS. Blocking was performed with 1% bovine serum albumin (BSA) overnight. After blocking, cells were incubated with primary antibody for 1 h, followed by three washes with PBS. Primary antibodies used were anti-rat-HA (clone 3F10; Roche), mouse anti-5E3 (kind gift from David Sibley, Washington University School of Medicine), and rabbit anti-histone 3 acetyl Lys9 (anti-H3K9ac; Millipore Sigma). Fluorophore-conjugated secondary antibodies (Alexa Fluor 488, Alexa Fluor 568; Invitrogen) at a dilution of 1:500 was added, and cells were incubated for 1 h. Washing was performed three times with PBS, and Hoechst 33342 DNA stain was added during the first wash. Coverslips were inverted and mounted on glass slides using Vectashield antifade mounting medium (Vector Laboratories). Images were captured using super-resolution structured illumination microscopy (SR-SIM) on a Zeiss ELYRA S1 microscope. Z-stack images were collected and processed using automatic SR-SIM parameters in ZEN 2011.

Cells grown on coverslips were fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with 50 µg/ml digitonin for 10 min or 0.1% Triton X-100 in PBS for 5 min, blocked with 0.1% (w/v) gelatin in PBS for 30 min, and then incubated overnight with primary antibodies against GFP (A-6455, Thermo Fisher Scientific) and LC3B (#2775, Cell Signaling Technology). After washing, cells were incubated with Alexa-Fluor-conjugated goat anti-rabbit-IgG and anti-mouse-IgG secondary antibodies (Thermo Fisher Scientific) for 60 min. Cells were imaged using a confocal laser-scanning microscope (FV1000, Olympus, Inc., Tokyo, Japan) with a UPlanSApo 100× NA 1.40 oil objective lens. z-projection stack images were acquired with z steps of 0.5 µm. SIM images were acquired on a Zeiss ELYRA S.1 microscope equipped with a Plan Apochromat oil-immersion objective (63×, 1.4 NA, Carl Zeiss). Image contrast and brightness were adjusted using Photoshop CS4 (Adobe).