Formaldehyde

After washing with PBS, N2a cells were fixed for 30 min with 4% formaldehyde (Polysciences, PA, USA) in PBS at room temperature. Cells were washed three times with PBS, followed by permeabilization with 0.1% Triton-X100 (Acros) in PBS for 10 min. After blocking for 20 min with 5% goat serum (Dako) and 1% BSA (Sigma-Aldrich) in PBS (blocking buffer), cells were incubated with the primary homemade antibody for endogenous NBEA (1/1,000), diluted in blocking buffer, for 1h. After three washes, cells were incubated with a goat secondary Alexa594- or Alexa488-conjugated antibody (Life technologies) and DAPI (1/10,000) for 1h. Finally the cells were washed and mounted on microscope slides using Vectashield mounting medium (Vector laboratories, Canada). Cells were analyzed using the Olympus Fluoview FV1000 confocal laser scanning microscope and a 63x immersion oil objective.
The tool ‘Z project’ in ImageJ software was used to make a 2D Z-projection of a confocal Z-stack. All confocal images shown are single confocal planes, unless otherwise specified.
Quantification of nuclear EGFP fluorescence intensity was performed using Cell Profiler cell analysis software [39 (link)]. At least 300 transfected cells were measured per condition.

Cells grown on glass coverslips were transfected with expression constructs using Lipofectamine 3000 (Life Technologies). After a 24-h incubation period, cells were fixed with 4% formaldehyde (Polysciences Inc., Warrington, PA, USA) in phosphate-buffered saline (PBS) for 15 min, permeabilised with 0.25% Triton X-100 in PBS for 15 min and incubated for 1 h with blocking solution containing 5% bovine serum albumin (Sigma-Aldrich) in PBS. Primary and secondary antibodies diluted in PBS were incubated for 1 h and 30 min, respectively. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Coverslips were mounted onto glass slides with Fluoromount aqueous mounting medium (Sigma-Aldrich). Images were acquired on a Nikon Ti Eclipse confocal laser-scanning microscope equipped with a 60X objective and analysed using ImageJ software [14 (link)]. All fluorescence images shown were chosen to best represent the average staining pattern for a particular experiment. In excess of 1,000 transfected cells were analysed for the experiments shown in Figs 1 and 2, and in excess of 200 transfected cells were analysed for each of the experiments shown in Figs 3–6.

To detect ROPs on PVM via semipermeabilization, confluent monolayers of HFFs were seeded onto coverslips and infected with ROP54HA_II parasites at the indicated time points. The samples were washed quickly with PBS and fixed in 4% formaldehyde (Polysciences) for 10 min at room temperature. The fixed coverslips were quenched with 100 mM glycine–PBS for 5 min at room temperature. The cells were permeabilized with either 0.002% digitonin–PBS (made fresh for each experiment) for 2.5 min at 4°C or 0.01% saponin–PBS for 30 min at room temperature. The samples were placed in blocking buffer (10% fetal calf serum [FCS]–PBS) for 30 min at room temperature to prevent nonspecific binding of the antibodies. Primary antibodies were diluted in blocking buffer (1:300 for MAb HA.11 [Covance], 1:300 for pAb ROP5 [Sibley], 1:100,000 for mouse SAG1 [DG52], and 1:100,000 for rabbit pAb SAG1) and used to probe the coverslips at room temperature for 1 h. The secondary antibodies Alexa 488-conjugated goat anti-mouse and Alexa 594-conjugated goat anti-rabbit (Invitrogen) were diluted at 1:2,000 in blocking buffer and added to the samples for incubation for 1 h (27 (link)). The coverslips were mounted in Vectashield (Vector Labs.) or ProLong Gold (Molecular Probes) and viewed with an Axio Imager.Z1 fluorescence microscope (Zeiss).

The following procedure was adapted from our previous work^{12 (link)}. Unless stated, about 10–20 organoids were collected into 1.7 ml Eppendorf tubes (Corning Inc). After aspirating the media, the organoids were fixed in 4% formaldehyde (Polysciences Inc, Warrington, PA) for 15 minutes at 4 °C and were washed 3 times with cold PBS. The organoids were permeabilized with 0.1% Tween-20 in PBS for 10 minutes at 4 °C and washed 3 times. Organoids were exposed to Protein Block (Dako Group, Troy, MI) for 1 hr at RT, and organoids incubated at 4 °C overnight in Antibody Diluent (Dako) solution containing primary antibodies, anti-beta Catenin (1:500, Abcam), anti-ZO-1 (1:1000, Millipore), and anti-Claudin-5 (1:500, Millipore). Organoids were subsequently washed 3 times and incubated with AF488 Goat anti-Mouse IgG (1:1000, Life Technologies), AF594 Goat Anti-Rabbit IgG (1:1000, Life Technologies) in Antibody Diluent (Dako) overnight at 4^oC. Nuclear staining was performed by incubating the organoids with DAPI (1:1000) in PBS for 10 minutes. The organoids were washed and were imaged using the Olympus Fluoview Fv10i (Olympus) laser scanning confocal microscope. Unless stated, at least three randomly selected organoids were imaged for each stain.

Embryos were fixed with 4% formaldehyde (Polysciences) for 10 min, permeabilized with 0.5% Triton X-100 (Sigma Aldrich) for 30 min, and then blocked with blocking solution (10% Fetal Bovine Serum (Hyclone), 0.1% Triton X-100) for 1 hr at room temperature, or overnight at 4°C. Primary Antibodies used were: mouse anti-CDX2 (Biogenex, CDX2-88), goat anti-SOX2 (Neuromics, GT15098), rabbit anti-PARD6B (Santa Cruz, sc-67393), rabbit anti-PARD6B (Novus Biologicals, NBP1-87337), mouse anti-PKCζ (Santa Cruz Biotechnology, sc-17781), rat anti-CDH1 (Sigma Aldrich, U3254), mouse anti-ZO1 (Thermo Fisher Scientific, 33–9100), mouse anti-YAP (Santa Cruz Biotechnology, sc101199), rabbit anti phospho-YAP (Cell Signaling Technologies, 4911), chicken anti-GFP (Aves, GFP-1020). Stains used were: Phallodin-633 (Invitrogen), DRAQ5 (Cell Signaling Technologies) and DAPI (Sigma Aldrich). Secondary antibodies conjugated to DyLight 488, Cy3 or Alexa Flour 647 fluorophores were obtained from Jackson ImmunoResearch. Embryos were imaged using an Olympus FluoView FV1000 Confocal Laser Scanning Microscope system with 20x UPlanFLN objective (0.5 NA) and 5x digital zoom. For each embryo, z-stacks were collected, with 5 µm intervals between optical sections. All embryos were imaged prior to knowledge of their genotypes.

smFISH was performed as described in the previous report with slight modification^{71 (link)}. Cells of WT (H1N2330), lem2Δ (H1N2324), and red1Δ (H1N2328) were pre-cultured in minimum medium (EMMG5S) for overnight at 30 °C, then transferred into rich medium (YES) and cultured for 16 hours. The cells (~1 × 10⁸) were mixed with one-tenth of meiosis-induced PSB1940 (YY548-13C) cells (~1 × 10⁷) as a positive control for smFISH, and then fixed with 4% formaldehyde (Polysciences) at 30 °C for 30 minutes. The cells were labeled with Quasar 570-labeled RNA probe set for sme2 (See Supplementary Table 4 in ref. ^{71 (link)} in detail), then mounted in ProLong Glass Antifade Mountant (Thermo Fisher Scientific).
The cells were observed using ×60 PlanApo N OSC oil-immersion objective lens (numerical aperture (NA) = 1.4, Olympus) on the DeltaVision Elite system (GE Healthcare) equipped with pco.edge 4.2 sCMOS camera (PCO). Chromatic shifts were corrected using Chromagnon software (v0.87) using a bleed-through fluorescence image as a reference^{72 (link)}. The images were denoised by the ND-safir program^{73 (link)}, deconvolved using the built-in SoftWoRx software (v7.0.0), and then projected by maximum-intensity projection. The brightness of the images was adjusted using the Fiji software^{74 (link)} for better visualization, without changing the gamma settings.