Anti goat alexa fluor 568

Livers were collected at 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 RT. Liver sections of 50 μm thickness were stained and analyzed as previously described [40 (link), 42 ]. 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 the determination of the size of P. berghei intrahepatic forms 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).

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).

Cells were fixed in 10% formaldehyde, treated with serum-free protein blocking solution (Dako), and then incubated overnight at 4 °C with antibodies against Scx (1:150, Abcam, Rabbit polyclonal), Tnmd (1:150, Santa Cruz Biotechnology, Goat Polyclonal), Runx2 (1:200, Abcam, Rabbit polyclonal), Sox9 (1:200 Santa Cruz Biotechnology, Rabbit polyclonal), PPARγ2 (1:200, Abcam, Rabbit polyclonal), or aggrecan (1:200, Abcam, Rabbit polyclonal). Cells were then rinsed in PBS. For Scx and Tnmd staining, cells were incubated with anti-rabbit Alexafluor 488 (1:1000, Invitrogen) or anti-goat Alexafluor 568 (1:1000, Invitrogen). For the other antibodies, cells were incubated for 30 minutes with anti-rabbit secondary antibody conjugated with horseradish peroxidase (Dako) followed by DAB chromagen (Vector Laboratories) for 2 minutes. Negative control sections were prepared using irrelevant isotype matched primary antibodies (Dako).