Live egesta samples were imaged on a Zeiss LSM 780 NLO confocal microscope system (Zeiss, Oberkochen, Germany). Images were taken on three separate channels, with excitation from a 405 nm Diode, Argon (488), and 633 nm HeNe. Emission was detected between 410–470 nm, 491–572 nm, and 647–722 nm, respectively. Simultaneously, using laser illumination, a transmitted light detector (T-PMT) acquired transmitted light images in bright field. Confocal z-stack images were processed for pseudocoloring and merged to create a maximum intensity projection using ZEN Black (Carl Zeiss AG) and ImageJ software. Separately, differential interference contrast (DIC) images were obtained using an Olympus Vanox-T AH2 microscope to image nematocysts.
Vanox t ah 2 microscope
Manufactured by Olympus
Sourced in Japan
The Vanox-T AH-2 is an optical microscope manufactured by Olympus. It is designed for routine observation and analysis of biological and material samples. The microscope features binocular observation, halogen illumination, and a range of magnification capabilities.
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4 protocols using vanox t ah 2 microscope
1
Multi-modal Imaging of Live Egesta
2
Peripheral Blood Smear Preparation and Staining
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Peripheral blood smears were prepared on microscope slides (Menzel-Gläser, Braunschweig, Germany) and stained with a May–Grünwald-Giemsa protocol. The staining procedure consisted of immersing the slides for 1.5 min in May–Grünwald's eosin-methylene blue solution (Merck, Darmstadt, Germany) and, after washing up the excess solution with water, staining the slides for 20 min with 10% Giemsa stain solution (Merck, Darmstadt, Germany). Furthermore, slides were left to dry for at least 30 min (Vives Corrons et al., 2004 (link)). Slides were visualized with an Olympus Vanox-T AH-2 microscope (Tokyo, Japan), equipped with an Olympus DP73 digital camera, using a 100 × oil objective (Olympus SPlan 100PL). Digital images were assessed using CellSens Entry software (V1.7, Olympus, Tokyo, Japan).
3
Cresyl Violet Staining and Neurodegeneration Analysis
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Slides were stained for 3 minutes with filtered 0.5% cresyl violet solution, rinsed in water for 5 minutes, and subsequently dehydrated and cover-slipped in Pertex medium. Photomicrographs for CA1, CA3, DG, PrL layers II and III, and dorsal tenia tecta (DTT) were obtained using a Vanox-T AH-2 microscope (Olympus). The number of neurons was counted manually, and neuronal density (a widely accepted metric for assessing neurodegeneration20 (link)) was measured in the various regions. In addition, in the PrL and DTT, where degenerating neurons could be easily identified as dark-stained neurons with shrunken cell bodies or presenting vacuolation, the percentage of cells with neurodegeneration was also quantified. Neuronal density and neurodegeneration were measured using Image J software, as shown in Appendix 1, Figure S2. The values for each animal were obtained by averaging the results for 3 sections.
4
Whole-Mount Embryo Imaging and Sectioning
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Images of whole‐fixed embryos were acquired with an Olympus SZH10 Stereo microscope equipped with a Q‐Imaging Retiga 2000 K camera, controlled using QCapturePro software. The embryos were positioned as desired in depression slides in PBS. Next, embryos were embedded in paraffin wax and sectioned sagittally at 10 μm using a microtome (Microm HM 315). They were mounted in 3:1 Canada Balsam (Merck KGaA, 8007‐47‐4): histoclear (HS‐202 HISTO‐CLEAR II, National Diagnostics) and imaged with an Olympus VANOX‐T AH2 microscope with 40x oil immersion objective (NA 0.7).
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