Axiocam 506

The PV/PNN stained slices were imaged at 5 × and 40 × on a Zeiss AxioImager.Z1 with Apotome (40 × only). We used Zeiss EC Plan-Neofluar 5 ×/0.16 M27 and Zeiss EC Plan-Neofluar 40x/0.75 M27 objectives, and imaged with a Zeiss Axiocam 506. From each sensory cortex, we collected between 1 and 10 non-overlapping images (number matched within each region to account for differences in the size of the brain region) for both layer 2/3 and 4, from each slice. Per animal, we used 4 slices per area, and per timepoint N = 3–6 each Ehmt1^+/+/Ehmt1^+/− mice, for a total of between 13 and 68 images (matched within one brain region) per genotype for each area and layer.
The NeuN/Gad65 stained slices were imaged at 5 × and 63 × on a Zeiss AxioImager.Z1 with Apotome unit (63 × only). We used Zeiss EC Plan-Neofluar 5x/0.16 M27 and Zeiss Plan-Apochromat 63 ×/1.4 Oil DIC M27 objectives, and imaged with a Zeiss Axiocam 506. We used one timepoint (P14) with N = 3/3 Ehmt1^+/+/Ehmt1^+/− mice, 4 slices per animal, and collected between 2 and 3 non-overlapping images of each auditory cortex layer 2/3 at 63×.

Worms were picked manually from mixed stage culture plates into 5μl of water on a 4% agarose slide and visualized under a microscope using DIC optics [49 ]. For the dauer staging, dauers were isolated from 2 week old plates that had been starved of food for at least a week and then visualized directly using DIC microscopy on a Zeiss Imager M2 microscope with a Zeiss Axiocam 506 mono camera. To determine the dauer recovery and number of molts, dauers were picked from the same plates onto fresh NGM plates supplemented with OP50 in groups of 5 and then incubated at 15°C. A plate was removed every 2 hours (upto 44 hours) and the worms on the plate were examined under 40x DIC microscopy to determine stage and changes in development between time points. High magnification images of the different stages was taken using a Zeiss Image Z1 with a Zeiss Axiocam 506 mono camera.

Tsetse flies (Glossina morsitans) were maintained at 27°C and 70% hygrometry in Roubaud cages, in groups of 50 male flies per cage. Pleomorphic trypanosome cell lines were maintained at 1 × 10⁵ cells/ml density in HMI-9 medium plus 10% FBS at 37°C with 5% CO₂. In vitro stumpy differentiation was induced in HMI-9, supplemented with 10% FBS without antibiotics, by adding 8-pCPT-2′-O-Me-5′-AMP (5 μM) (BioLog-Life Science Institut) to the culture 48 h before fly infection (Laxman et al., 2006 (link)). On the day of infection, trypanosomes were resuspended at 10⁶ cells per ml in SDM79 with no antibiotics supplemented with 10 mM glutathione prior infection (MacLeod et al., 2007 (link)). Flies were fed on infected media through a silicone membrane and maintained until dissection by feeding three times per week on sheep’s blood in heparin. Flies were starved for 2 days before dissection at day 28. Imaging was carried out using a ZEISS Imager 72 epifluorescence microscope with an Axiocam 506 mono camera. Single images (for DIC) or multichannel stacks (for fluorescence) of images every 0.24 µm were acquired. When maximum intensity Z-projections are presented, they were generated using Fiji (Schlindelin et al., 2012 (link)).