C11440 digital camera

PC-3U cells were treated, or not, with TGFβ for 30 min, fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. For brightfield PLA analysis, the tissue sections were pretreated with deparaffinization, retrieval and permeabilization. In situ proximity ligation assay was performed according to the manufacturer's instructions with the Duolink Detection Kit (Sigma). Photomicrographs for immunofluorescence were taken with an AX10 microscope (Carl Zeiss) using a 40 × 0.95/NA objective lens (Carl Zeiss). The images were acquired with Zen 2 pro software at room temperature. The camera was a Hamamatsu Digital Camera C11440. Images for brightfield were acquired with Pannoramic 250 Flash.

Formalin-fixed tumors were dehydrated and paraffin embedded according to standard procedures. 5-μm slices were cut using a microtome, rehydrated, and subjected to antigen unmasking by heating at 95°C for 30 minutes with a commercially available antigen unmasking solution (Citra). Samples were incubated with primary antibodies, washed, and incubated with fluorescent labelled secondary antibodies. Nuclei were stained through 4′,6-diamidin-2-fenilindolo (DAPI) incubation and slices were mounted. Immages of samples were acquired using a fluorescence microscopy Nikon Eclipse Ni, equipped with a digital camera (Hamamatsu Digital Camera C11440).

Retinal sections were permeabilized in 0.05% (v/v) TritonX-100 for 1 h and then were blocked with 10% (v/v) donkey serum (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Retinal sections were incubated with primary antibodies (Table 3) at 4 °C overnight in a humidity chamber. After washing with PBS × 1 for 3 times, retinal sections were then incubated for 1 h at room temperature with fluorescent secondary antibodies (Table 3). Some sections were continually incubated in TUNEL mixed solution for another 1 h. After washing with PBS × 1 for 3 times, sections were counter-stained with Hoechst 33258 and mounted in 10% (v/v) Mowiol^® solution (Polysciences, Eppelheim, Germany).
Retina whole mounts were permeabilized 0.2% (v/v) TritonX-100 for 30 min. The retina whole mounts were incubated in conical small wells and transferred with 3 mL pipette. The staining steps were the same as those of immunoistochemistry section. Finally, the retinal whole mounts were mounted on microscope slides and covered with thinner and smaller cover slides in 10% (v/v) Mowiol^® solution. Images of fluorescent sections and whole mounts were obtained using an epifluorescence Olympus BX60 microscope connected to a Hamamatsu C11440 digital camera.

The transgenic P. berghei parasite line pv5-tag-GFP^PV (27 (link)) was imaged live using an AxioImager Z2 epifluorescence microscope equipped with an AxioCam MR3 camera (Zeiss). For mechanical parasite expansion, 1 to 2 µL of infected blood was incubated under a 22 × 40-mm coverslip for several minutes until erythrocyte lysis became apparent. P. falciparum parasites were imaged on an Eclipse Ni light microscope (Nikon) fitted with a C11440 digital camera (Hamamatsu). Immunofluorescence analysis was performed with P. falciparum pv5-3xHA:loxP parasites that were fixed in 4% formaldehyde using rat anti-HA (1:500; Sigma Aldrich) and rabbit anti-SERA5 (1:500) (55 (link)) primary antibodies in combination with appropriate fluorophore-coupled secondary antibodies (1:1,000; Thermo Fisher Scientific). Staining with Lysosensor blue DND-167 (Thermo Fisher Scientific), BODIPY 581/591 C11 (Image-iT Lipid Peroxidation Kit, Thermo Fisher Scientific), and CellROX Green (Thermo Fisher Scientific) was performed according to the manufacturer’s instructions.