Lead citrate solution

Mice were sacrificed and transcardially perfused using first PBS and subsequently 4% (w/v) PFA. The gut was dissected and mouse intestine samples with a size of approx. 1 mm³ were incubated in 0.1 M sodium cacodylate buffer (pH 7.2), 2.5% (v/v) glutaraldehyde, 4% (w/v) PFA for 2 h and washed thrice with 0.1 M sodium cacodylate buffer. Increases in contrast were achieved by incubation with 1% (w/v) OsO₄ for 1 h. After washing with 0.1 M sodium cacodylate buffer, samples were dehydrated by increasing ethanol concentrations (30, 50, 70, 80, 90 and 100% (v/v)) and stained with uranylacetate (2% (w/v) uranylacetate in 50% (v/v) ethanol). The samples were then embedded in araldite resin for 48 h at 60 °C.
Ultrathin sectioning of embedded samples was performed using a LKB 8800 A Ultratome III (LKB Produkter AB). 60 nm thin sections were collected onto formvar-coated grids and stained with lead citrate solution (Electron Microscopy Sciences) for 2 min.
Samples were blinded and images were taken using an EM902A transmission electron microscope (Carl Zeiss AG) operated at 80 keV. Images were recorded digitally using a 1 k FastScan CCD camera (TVIPS camera and software).

All the routinely used biochemicals were of molecular biology grade and procured from MERCK or similar sources unless otherwise stated. Cell culture reagents were from Lonza and Thermo Fisher Scientific. Restriction enzymes and DNA modifying enzymes were from New England Biolabs and Thermo Fisher Scientific. DNA isolation kits were from QIAGEN and MACHEREY-NAGEL. ProLong Diamond Antifade Mountant, Hoechst, DAPI, SuperSignal Chemiluminescent substrate and 2’,7’-dichlorodihydrofluorescein diacetate acetyl ester (H₂DCFDA) were from Thermo Fisher Scientific. Secondary antibodies were from Thermo Fisher Scientific and Jackson ImmunoResearch. Primary antibodies were from Cell Signalling Technologies, Thermo Fisher Scientific, MERCK and ChromoTek. WR99210 was a kind gift from David Jacobus (Jacobus Pharmaceutical, Princeton, U.S.A.). Protease inhibitor cocktail, trimethoprim and N-acetylglucosamine (GlcNAc) were from MERCK. Glutaraldehyde, formaldehyde, osmium tetroxide, lead citrate solution, and sodium cacodylate buffer were from Electron Microscopy Sciences. Uranyl acetate was from Loba Chemie and epoxy resin constituents were from TED PELLA. P. falciparum 3D7 and D10 strains were obtained from the Malaria Research and Reagent Reference Resource centre (MR4). All the plastic ware was from standard manufacturers such as Corning Inc, Nalgene, Thermo Fisher Scientific and Tarsons.