Dm5000b upright microscope

One female of Caligusdussumieri (NHMUK 2022.189–197) was examined using CLSM. The specimen was stained overnight in a saturated solution of Congo Red in 100% ethanol, then rinsed in distilled water until no Congo Red could be seen diffusing and prepared as a temporary mount in a 50% solution of glycerine and distilled water on a glass slide under a coverslip. The specimen was examined using a Leica TCS SP5, equipped with a Leica DM5000 B upright microscope and the Leica Application Suite Advanced Fluorescence software LAS AF 2.2.1. (Leica, Wetzlar, Germany). We used a 561 nm excitation wavelength from a DPSS 10 mW 561 nm laser set at 80% power and collected the emitted fluorescence in two channels: 570–630 nm artificially coloured green and 630–715 nm artificially coloured red. A series of image stacks were collected and the final images were obtained by maximum projection of the overlaid channels using the same Leica software. For the full-body dorsal and ventral images, multiple fields of view were combined using Adobe Photoshop v.25.1.

Bacterial cell morphology was examined to assess if the peptides cause filamentation of B. pseudomallei cells, indicative of inhibition of in vivo DNA synthesis (Alfred et al., 2013 (link)). About 5 × 10⁵ CFU/ml bacterial suspension was exposed to each peptide at 8× MIC₉₀. Bacterial cells treated with 1× PBS were used as the negative control. After 1 h incubation at 37°C, an aliquot of the reaction mixture was spotted onto a microscope slide, air dried and stained with crystal violet for 1 min. Excess stain was subsequently rinsed off using distilled water and air-dried. All samples were observed using the Leica DM5000B upright microscope (1000× magnification). Each image was captured using identical settings and the experiment was performed in triplicate.

Tibialis anterior muscles were cut across the midbelly and 10 µm sections transferred onto glass slides using a Microm HM 550 Cryostat (Microm International, Walldorf, Gemany). Tissue sections were fixed with 4% paraformaldehyde for ten minutes, then incubated with wheat germ agglutinin conjugated to Alexa Fluor™ 594 (Thermo Fisher Scientific) for 1 hour at room temperature. Whole diaphragm muscles were cryosectioned and fixed as above and stained for myosin heavy chain type I (clone BA-D5, Developmental Studies Hybridoma Bank), myosin heavy chain type IIa (clone SC-71, Developmental Studies Hybridoma Bank) and wheat germ agglutinin conjugated to Alexa Fluor™ 594 (Thermo Fisher Scientific). Secondary antibodies used for MHC type I and MHC type IIa were Alexa Fluor™ 350 and 488, respectively. TA and diaphragm sections were imaged on a Leica DM5000 B upright microscope, and muscle fiber cross-sectional area was measured using ImageJ. A minimum of 250 fibers was measured per muscle.

Whole-mount embryos stained by NBT/BCIP were mounted in 80% glycerol. Sections of adult spinal cord were mounted in a solution of Mowiol. Embryos were imaged using a Nikon AZ100M macroscope and a Leica DM5000 B Upright microscope. Adult spinal cord sections were imaged using a Nikon AZ100M macroscope. To quantify pkd2l1⁺ and GABA⁺ cells, stained embryos were imaged using the Fixed Stage microscope AxioExaminer Z1 equipped with a 20× water immersion objective and a Yokogawa CSU-X1 spinning disk unit (n = 13 embryos for pkd2l1 and n = 10 for GABA). To quantify the overlap of GFP with pkd2l1 FISH in the Tg(nkx2.2a:mEGFP) and the Tg(olig2:EGFP) transgenic embryos, 50 μm-thick sections were analyzed on an Olympus FV1000 confocal microscope equipped with a 40× water immersion objective using 405, 473, and 543 nm laser lines. Images were processed using Fiji (Schindelin et al., 2012 (link)) and Adobe Illustrator (Adobe Systems, Mountain View, CA, USA) software.