Axio imager m2 fluorescence microscope

For fluorescence microscopy, cells were grown at 28°C in LB medium to an OD₆₀₀ of 0.2–0.5 (unless otherwise indicated). Fluorescence images were obtained with the AxioImager M2 fluorescence microscope, equipped with digital camera AxioCam MRm and AxioVision Rel 4.8 software for image processing and an EC Plan-NEOFLUAR 100X/1.3 objective (Carl Zeiss, Göttingen, Germany). The applied filter sets were the Filter set 49 (G 365, FT 395, BP 445/50; Carl Zeiss) for DAPI detection, the Filter set 38 (BP 470/40, FT 495, BP 525/50; Carl Zeiss) for GFP detection, and Filter set 43 (BP 545/25, FT 570, BP 605/70) for Nile Red visualization. The overlays of fluorescent and phase-contrast images were prepared for presentation with ImageJ 1.49p (National Institutes of Health, Bethesda, USA). Images were taken with an exposure time of 1 s for the GFP constructs (except for Eno-GFP, exposure time 500 ms), 500 ms for the membrane stain Nile Red, and 100 ms for the DNA stain DAPI.

For the immunofluorescence analyses, cells cultured on coverslips (Matsunami) were fixed with −20°C methanol for 7 min, or 4% PFA at room temperature for 30 min. Fixed cells were incubated with blocking buffer (1% bovine serum albumin in PBS containing 0.05% Triton X-100) for 30 min at room temperature. The cells were then incubated with primary antibodies (see below) in the blocking buffer for 1 h in a humid chamber. After washing with PBS, the cells were incubated with secondary antibodies and Hoechst 33258 (DOJINDO, 1:3000–1:5000) in the blocking buffer for 30 min, followed by a final wash with PBS. The coverslips were mounted onto glass slides (Matsunami) using ProLong Gold Antifade Mountant (Molecular Probes), with the cell side down. Images were acquired as z-stacks with an AxioImager M2 fluorescence microscope (Carl Zeiss) equipped with a 63×/1.4 or 40×/1.3 NA objective lens. The fluorescence intensity of centrosomal proteins was measured from the maximum projection images using FIJI (National Institutes of Health, Bethesda, MD, USA). The mean fluorescence intensity of a fixed-size area around the centrosome was calculated. The measurement was corrected for background intensity by subtracting the cytoplasmic signal within the same size area near the centrosome.

In situ hybridizations were performed on chicken (HH stage 14–26) and mouse (E9.5–10.5) embryonic spinal cords. 3’UTR probes were designed using http://primer3plus.com and verified for specificity to the gene of interest using http://www.ncbi.nlm.nih.gov/tools/primer-blast/.Chicken and mouse primer sequences used to make in situ hybridization probes can be found in Supplementary file 2 and 3 respectively. Probes were made using a DIG RNA labeling kit (Roche, Indiananapolis, Indiana). Images were collected on a Carl Zeiss AxioImager M2 fluorescence microscope with an Apotome attachment.

Parasites in the two different life stages, epimastigotes and trypomastigotes, were collected by centrifugation (5000× g for 30 min at 4 °C), washed twice with PBS, fixed with paraformaldehyde (4%) in suspension for 5 min and washed 3× in PBS. The parasites were placed onto a glass slide and allowed to air-dry overnight. Next, cells were blocked with 1% casein in PBS (pH 7.4) for 15 min at 37 °C. Afterward, parasites were incubated with rabbit anti-Tc/NICT-1 sera (1:100) for 2 h at 37 °C. Slides were washed in PBS and then incubated with TRITC-labeled anti-rabIgG (1:400) for 1 h at 37 °C. Additionally, anti-β-tubulin (1:400) was used to probe parasites’ microtubules. Following this, parasites were washed in PBS, stained with DAPI and mounted in DABCO solution. An Axio Imager M2 fluorescence microscope (Carl Zeiss) was used to collect images.