Livers were collected at either 6, 12, 24 or 46 h after P. berghei sporozoite inoculation, and fixed in 4% (v/v) paraformaldehyde (PFA) (SantaCruz Biotechnology, Dallas, TX, USA) for at least 12 h at room temperature (RT). Liver sections of 50 μm thickness were stained and analyzed as previously described [79 (link), 81 (link)]. Briefly, slices were incubated in permeabilization/blocking solution containing 1% (w/v) bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and 0.5% (v/v) Triton-X100 in PBS (Sigma-Aldrich) at RT for 1 h, followed by a 2 h incubation at RT with an anti-UIS4 antibody (goat, homemade; dilution 1:500). Liver sections were further incubated for 1 hour in a 1:500 dilution of anti-GFP-Alexa 488 antibody (Invitrogen, Carlsbad, CA, USA) and anti-goat Alexa-Fluor 568 (Invitrogen, Carlsbad, CA, USA) in the presence of a 1:1,000 dilution of Hoechst 33342 (Invitrogen, Carlsbad, CA, USA). After washing, liver sections were mounted on microscope slides with Fluoromount (SouthernBiotech, Birmingham, AL, USA). Widefield images for P. berghei intrahepatic forms’ size determination were acquired employing a Zeiss Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany). Confocal images were acquired using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany). Images were processed with the ImageJ software (version 1.47, NIH, Bethesda, MD, USA).
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