Mrm camera

Whole mount immunofluorescence of seminiferous tubules was performed as previously described^{43 (link)}. All images were captured with a Zeiss Imager Z1 microscope using a Zeiss MRm camera. Two testes at P7 and two testes at P60 were examined for this study.

Fluorescently labeled parasites were visualized under an AxioImager ZI inverted fluorescence microscope fitted with an Mrm camera (Carl Zeiss) for microscopic analysis. Images were acquired with a preset exposure time using AxioVision 4.2.8 software, z-stacks were taken for each image, and an appropriate scan was used for illustration in figures. For 3D reconstruction, the z-stack images from the 3D7 and R3_S804A parasites were subjected to the 3D module of the AxioVision 4.2.8 software. The surface interface was used for 3D construction. Pearson’s correlation coefficient was calculated using Zen software (Zeiss).

All procedures were performed by an experienced operator who was blinded to patient characteristics. In brief, the cells were blocked and permeabilized with 3% bovine serum albumin (BSA) (Roth) and 0.1% Triton X-100 (Fluka) in PBS for 30 min. Primary antibodies, including anti-pan-cytokeratin-Alexa 488 (CK4, 5, 6, 8, 10, 13, 18) (C-11) (Exbio), anti-CK19-Alexa 488 (A53-B/A2) (Exbio), anti-CK7- fluorescein isothiocyanate (LP5K) (FITC) (Millipore), anti-EpCAM- fluorescein isothiocyanate (FITC) (HEA125) (Acris), and anti-CD45- Alexa 647 (MEM-28, Exbio), were added for 30 min. The DC01 was then rinsed three times with 3 mL of PBS, and the nuclei were counterstained with Hoechst 33342 (Invitrogen). CTCs were identified using a Zeiss Axio imager fluorescent microscope with a 20× objective. Fluorescent images were recorded with a Zeiss MRm camera. A cell was considered to be a CTC if it was positively stained for cytokeratin and/or EpCAM, it was negative for CD45, and certain morphological criteria for tumor cells were met: the presence of a nucleus with a round or ellipsoid shape and a cell size ranging from 4 to 50 μm. Leukocytes were defined as nucleated (Hoechst-positive), CD45-positive, and cytokeratin and/or EpCAM-negative cells, and were not counted.

At the end of the study hearts were excised and representative pieces of left ventricles were fixed in 4% formaldehyde after rinsing with saline. For analysis of cardiac fibrosis, 5-μm sections of paraffin embedded hearts were stained with picrosirius red. Stained sections were digitalized with a SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany). Quantification was made with Tissue IA software (Leica Biosystems, Dublin, Ireland). Area occupied by interstitial fibrosis was expressed as a percentage of total myocardial area. To quantify transverse cardiomyocyte area and capillary density, cryosections were stained with Wheat germ agglutinin (WGA) as a membrane marker, and with biotinylated-Isolectine B4 as an endothelial marker. Mounted slides were observed with an Axioimager Z1/Apotome microscope equipped with a MRM camera (Zeiss, Germany). The data analysis was performed with Axiovision software (Zeiss, Germany). A minimum of 40 to 100 cells from 9 sections were measured in each heart. Other heart cryosections were incubated with antibodies raised against the monocyte marker CD11b and the lymphocytic marker CD45 (rat anti-CD11b and rat anti-CD45, both from BD Bioscience, San Jose, CA). Slides were scanned with a MIRAX Scanner (Zeiss, Germany) and analyzed with FRIDA software (Johns Hopkins University, Baltimore, MD). All analyses were performed on a blinded basis.

Transversal sections were imaged on a Zeiss Imager.M2 with an ApoTome.2 module using HXP 200C illumination. Immunostained sections of embryos of different stages were imaged with 40x Plan-Apochromat, 0.95 Korr (except Figures 5B and 20x Plan-Apochromat, N/A 0.8). Whole mount embryos were imaged on same microscope with 5x (EC Plan NeoFluar) or 10x (Plan-Apochromat, N/A 0.45) objective with Axiocam506 mono (fluorescence) and color (DIC/brightfield) cameras (except Figure 5B, imaged on Olympus MVX10 with Zeiss MRm camera). Figure 1B’ was imaged on Zeiss 710 inverted confocal microscope with LD C-Apochromat 40x/1.1 W Korr UV-VIS-IR objective.