ScalesSQ tissue clearing was performed as previously described by Hama et al., 2015 (link). Briefly, we incubated organoids in ScaleSQ (22.5% (w/v)) D-sorbitol (Sigma) and 9.1 M urea (Sigma) for 2 hr at 37°C. Next, we exchanged the solution to ScaleS4 (40% (w/v)) D-sorbitol, 10% (w/v) glycerine (Roth), and 15–25% (v/v) DMSO (Sigma) also at 37°C. ScalseS4 was renewed after 2 hr and the organoids were maintained at 37°C until analysis. This is critical, as the clearing effect decreases visibly after 1 hr at room temperature.
Glycerine
Manufactured by Carl Roth
Sourced in Germany
Glycerine is a colorless, odorless, and viscous liquid. It is a chemical compound with the molecular formula C3H8O3. Glycerine is a versatile substance commonly used in various industries, including pharmaceuticals, cosmetics, and food production, due to its lubricating, humectant, and sweetening properties.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Carl Roth (2 mentions)
Triton x 100, by Merck Group (2 mentions)
D sorbitol, by Merck Group (1 mentions)
D sorbitol, by Carl Roth (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Lsm 700 microscope, by Zeiss (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Genejet genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
Lb media, by Carl Roth (1 mentions)
Luna universal qpcr master mix, by New England Biolabs (1 mentions)
Nanophotometer, by Implen (1 mentions)
5 protocols using glycerine
1
ScalesSQ Tissue Clearing Protocol
2
Autofluorescence-Based Cuticle Sclerotization
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Hind wings were cut off and embedded in glycerine (≥99.5%, Carl Roth GmbH and Co. KG, Karlsruhe, Germany) between a glass slide and a cover slip. The specimens were then visualized with a Zeiss LSM 700 microscope (Carl Zeiss Microscopy, Jena, Germany) equipped with four stable solid‐state lasers with wavelengths of 405, 488, 555, 639 nm. The obtained images were used to determine the level of the sclerotization of the hooks, based on the autofluorescence of their materials: i) when excited with a stable solid‐state laser with a wavelength of 405 nm and visualized using a bandpass emission filter transmitting 420–480 nm, the soft nonsclerotized cuticle emits blue light; ii) when excited with a stable solid‐state laser with a wavelength of 488 nm and visualized using a longpass emission filter transmitting ≥490 nm, the less‐sclerotized cuticle emits green light; iii) when excited with stable solid‐state lasers with wavelengths of 555 and 639 nm and visualized using longpass emission filters transmitting ≥560 and ≥640 nm, respectively, the sclerotized cuticle emits red light.[20
] In total, seven wings were examined.
] In total, seven wings were examined.
3
Antennal Dissection and Microscopy
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Prior to dissection, the animals were anaesthetised with CO2. Dissection was performed in phosphate buffer solution (PBS) (Carl Roth GmbH & Co KG, Karlsruhe, Germany). The specimens were briefly subjected to small amounts of Triton X-100 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), to remove air bubbles trapped on the surface by decreasing water surface tension. Triton X-100 then was washed repeatedly with the PBS to fully remove traces of Triton X-100. Microscopical observations were made after transfer of antennae or antennal fragments to glycerine (Carl Roth GmbH & Co. KG, Karlsruhe, Germany).
4
Antenna Morphology Analysis in Insects
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Animals were deeply anaesthetized with CO2, and dissected in PBS (Carl Roth GmbH & Co KG, Karlsruhe, Germany). The antennae were treated with the surfactant Triton X-100 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), to ensure wetting of the entire surface, a necessary step as the fibrillae of plumose antennae easily trap air bubbles. Triton X-100 was washed off in triple steps with PBS. Antennae or their parts were transferred to glycerine (Carl Roth GmbH & Co. KG, Karlsruhe, Germany), which is a suitable medium as it has a similar refractive index to glass [16 (link)].
In the present study, only males are included. There are three main reasons for this: (1) due to strong structural sexual dimorphisms, morphological sex comparisons are difficult. (2) While acoustics is important to both sexes (e.g. [30 (link),32 (link)]), the male antenna is the most studied with respect to their acoustic response, lending itself to easier comparison. (3) CLSM as relative method benefits more from comparing two separate, but structurally similar objects. Four individuals of each species were used for the present study, not all of which were CLSM-imaged, but it was confirmed the structures imaged are typical, either under the CLSM Zeiss LSM 700 (Carl Zeiss Microscopy GmbH, Jena, Germany) or a fluorescence microscope (Zeiss Axioplan).
In the present study, only males are included. There are three main reasons for this: (1) due to strong structural sexual dimorphisms, morphological sex comparisons are difficult. (2) While acoustics is important to both sexes (e.g. [30 (link),32 (link)]), the male antenna is the most studied with respect to their acoustic response, lending itself to easier comparison. (3) CLSM as relative method benefits more from comparing two separate, but structurally similar objects. Four individuals of each species were used for the present study, not all of which were CLSM-imaged, but it was confirmed the structures imaged are typical, either under the CLSM Zeiss LSM 700 (Carl Zeiss Microscopy GmbH, Jena, Germany) or a fluorescence microscope (Zeiss Axioplan).
5
Quantitative Detection of Pseudomonas aeruginosa
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All chemicals, unless stated otherwise, were purchased from Sigma-Aldrich, subsidiary of Merck (Darmstadt, Germany). LB media, agar agar, diethylamine, glycerine, PD-tips, and sodium hypochlorite were purchased from Carl Roth (Karlsruhe, Germany). Polyglycerol-3-glycidyl ether (CL9) was obtained from ipox chemicals (Laupheim, Germany). All oligonucleotides were synthesized by Eurofins Genomics (Ebersberg, Germany), purchased in lyophilized form and dissolved in nuclease-free water. Primers for detection of the P. aeruginosa regA gene were designed in house. For genomic DNA extraction, the extraction kit GeneJET Genomic DNA Purification Kit by Thermo Scientific (Waltham, USA) was used. Photometric measurements were performed on a NanoPhotometer by Implen (Munich, Germany). For qPCR, the Luna® Universal qPCR Master Mix by New England Biolabs (Ipswitch, USA) was used. For all experiments, ultrapure water was used, unless stated otherwise. Nuclease-free water was purchased from Invitrogen AG (Carlsbad, USA). A bacterial isolate of Pseudomonas aeruginosa (ATCC27853) was received from the Bavarian Health and Food Safety Authority. Viable bacteria were handled in laboratories approved for biosafety level 2.
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