Dm5000 b

Fixed PSM explant tissue was proteinase K (Roche) treated and fixed (4% formaldehyde in PBS, 2 mM EGTA, 0.1% glutaraldehyde [Sigma]) prior to washing in PBST. Explants were then blocked in 2% bovine serum albumin (BSA) in PBST for 2 hr at room temperature. Anti-phospho histone-H3 antibody (Upstate) was then added at 10 μg/ml and the samples incubated at 4°C overnight. Specimens were then washed for 10 hr in a minimum of three changes of PBST before the Alexa-fluor488 conjugated mouse anti-rabbit antibody (Invitrogen) was added at 2 μg/ml in PBST and left overnight at 4°C. Samples were then washed for 10 hr in PBST with a minimum of 3 changes of solution. Explants were then mounted on SuperFrost microscope slides (VWR) using Hydromount (National Diagnostics) and imaged using either a compound fluorescence microscope (Leica DM5000 B) or a Zeiss 710 confocal microscope. Images were quantified using Volocity v6.0.1 imaging software (Perkin Elmer) and either t-tests or Mann–Whitney tests performed using SigmaPlot v12.0 software.

C56Bl6/J mice (7 days to 4 month old) were used to verify scRNA-seq candidate gene expression. Dissected mouse mandibles were fixed in 4% paraformaldehyde pH 7.4 overnight, decalcified in 10% EDTA for 7 days at +4 °C for 7 days (Foxd1, Sfrp2) or in 0.5 M EDTA at +4 °C for 20 days (Gjb3, Krt15, and Igfbp5). All samples were embedded in paraffin and sectioned at 7 μm. Tissue were subsequently processed using the RNAscope multiplex fluorescent detection reagents v2 (ACD, 323110) (Foxd1, Sfrp2) or RNAscope 2.5 HD Assay-RED detection kit (ACD, 322350, 322360) (Gjb3 (508841), Igfbp5 (425731), Smpd3 (815591), Dkk1 (402521), Wnt6 (401111), Wisp1 (501921), Nupr1 (434811), Syt6 (449641), Tac1 (410351)) according to the manufacturer’s instructions. Notably, slides were boiled in the target retrieval buffer and incubated in Protease Plus solution at 40 °C for 15 min before probes were incubated at 40 °C for 2 h. The following probes were used: Foxd1 (495501), Gjb3 (508841), Igfbp5 (425731), Sfrp2 (400381). Samples were counterstained either with DAPI for 30 s (Sfrp2 and Foxd1) or with Hematoxylin Gills #2 (20% dilution) for 15 s, followed by 10 s in ammonium hydroxide. Imaging was performed using a Leica DM5000 B (Gjb3, Igfbp5) or Zeiss LSM880 laser scanning confocal microscope (Foxd1, Sfrp2).

To detect CSP expression in PBANKA_072090 null mutant oocysts, parasites at day 14 p.i. were stained with 3D11 mouse anti-PbCSP [82 (link)] (10 μg/mL) as primary antibody and goat anti-mouse IgG-Cy™3 (Jackson ImmunoResearch Laboratories, Inc., #115-166-003; 1:400) as secondary antibody. In these IFAs, samples were fixed with 4% PFA/PBS for 10 min at RT and simultaneously permeabilized and blocked for 1 h at RT with a mixture of 0.5% TritonX-100/PBS and 1% BSA/PBS. All antibody incubations were done in permeabilizing/blocking solution for 1 h at RT and 5 ug/mL of Hoechst-33342/PBS was used to stain nuclei. Images were taken with a Leica DM5000B or Zeiss Axiovert 200 M fluorescence microscope and processed using ImageJ 1.47n software (imagej.nih.gov/ij).