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30 protocols using las 4

1

Imaging and Preservation of Insect Specimens

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Specimens were either pinned or point mounted on black, acid-free cards for examination (using a Leica M205C stereomicroscope with LED light source), photography, and long-term preservation. Images were taken using the Leica LAS 4.4 system which comprised a Leica Z16 microscope with a Leica DFC450 Camera with a 0.63× video objective attached. The imaging process, using an automated Z-stepper, was managed using the Leica Application Suite V 4.4 software installed on a desktop computer. Diffused lighting was achieved using a Leica Dome. Images of the types held in Musée Royal de l’Afrique Centrale, Tervuren (RMCA) were kindly made available by Stéphane Hanot and Arnaud Henrard and those in the Muséum national d’Historie naturelle, Paris (MNHN) were kindly made available by Agnièle Touret-Alby. All images presented in this paper are available at www.waspweb.org (van Noort 2020 ).
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2

Histological Analysis of Testicular Abnormalities

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Testes from E19.5 embryos of the F1 generation and P200 male mice of the F1 and F2 generations were fixed overnight at 4°C in Bouin’s solution with rotation, and then were embedded in paraffin. Five-micrometer-thick serial sections were cut and mounted on 3-aminopropyltriethoxysilane (APS)-coated slides (Matsunami APS-01); the mounted sections were deparaffinized, stained with hematoxylin and eosin, and examined using an optical microscope (Leica). Digital images were obtained using a LAS4.4 (Leica), and abnormal tubules were determined by the histological criteria described in the Results section. To determine the percentage of abnormal tubules, the numbers of abnormal tubules and total tubules were counted in each testicular section. Four sections, each 100 μm apart in a given testis, were counted.
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3

Quantitative Analysis of 17β-HSD6 Immunoexpression

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The analysis of the immunofluorescence was performed using a DFC 550 Camera (Leica) attached to a BM4000 B LED microscope (Leica), and the Leica Application Suite software (LAS 4.3, Leica). The measurement of 17β-HSD6 immunoexpression was performed in two non-serial testicular sections per animal from the CG, B12G, CMTG and CMT/B12G (n = 5/group). In the sections, the 17β-HSD6 immunofluorescence was measured in a standardized interstitial tissue area of 120,000 μm2 per animal at x220, and the immunofluorescent area per μm2 of interstitial tissue was obtained. All the parameters of the software, including threshold adjustment and color range—hue, saturation and intensity (LAS 4.3, Leica) were calibrated and standardized for each image analyzed.
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4

Quantitative Immunofluorescence Analysis of Epididymal Proteins

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The analysis of the immunofluorescence was performed using a DFC 550 Camera (Leica) attached to a BM4000 B LED microscope (Leica), and the Leica Application Suite software (LAS 4.3, Leica) .
The measurement of AR, KPNA and SHBG immunoexpression was performed in the proximal region of four non-serial sections of the cauda epididymidis per animal. In the AR and KPNA immunolabeled sections, a standardized total epithelial area of 50,000 µm 2 was measured. In this area, the nuclear and cytoplasmic AR immunofluorescent areas as well as KPNA immunofluorescent area were measured, and the area per µm 2 of epithelium was obtained. The AR immunofluorescent nuclear/ cytoplasmic ratio was also calculated. SHBG immunofluorescent area was measured in a standardized total connective tissue area of 560,000 µm 2 per animal, and the immunofluorescent area/µm 2 of connective tissue was calculated.
During each immunoreaction analyzed in the sections from CG and CMTG, all the parameters of the software, including threshold adjustment and color range -hue, saturation and intensity (LAS 4.3, Leica) -were rigorously calibrated/ standardized for each image analyzed.
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5

Dissection and Imaging of Cockroach Anatomy

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Ten-day-old adult female cockroaches were dissected under an Olympus SZ61 stereo microscope. Individuals were anesthetized using a CO2 stream, and cleaned with different solutions: bleach 10%, ethanol 70%, and two passages in the type II sterile water. Then, they were fixed with pins in a dorsal position on a silicone plate. They were ventrally opened via a longitudinal incision in the abdomen, and hindguts and fat bodies were extracted and gently washed with Krebs–Ringer Bicarbonate Buffer (Sigma-Aldrich, Burlington, MA, USA). Hindguts were immediately fixed for electron microscopy, and fat body samples were frozen in liquid nitrogen and stored at −80 °C until use for DNA extraction and acid uric determination. For in vivo visualization, ventrally-opened individuals were photographed using a Leica Z16 microscope equipped with a CF500 camera and LAS 4.9 (Leica, Wetzlar, Germany) software. Z-stacks, followed by extended depth of field application, were extensively used to produce final images.
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6

Identification of Neophyllaphis Aphids

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Individuals preserved in 70% ethanol were cleared using KOH and acetic acid and mounted in slides with Canada balsam. The morphological identification of the specimens was done using a Leica Z16 microscope. We measured structures with taxonomic value and used the keys from Miller and Halbert (2014) (link) and Blackman and Eastop (2019) to identify species of Neophyllaphis. The photographs were taken with a Leica Z16 microscope, equipped with a CF500 camera and LAS 4.9 (Leica) image capture. Mounted specimens were deposited at the aphid collection of the Instituto de Biología Integrativa de Sistemas (Centro Mixto Universidad de Valencia-CSIC, Spain) and in the Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica.
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7

Liposomal Diffusion Characterization via Tracking

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The in vitro diffusion properties of various liposomal preparations were characterized by multiple-particle tracking technology. This method allows simultaneous measurement of trajectories for hundreds of individual particles, facilitating relatively high throughput measurements30 (link). Poly(ethylene oxide) (PEO) hydrogels was used as the diffusion medium31 (link). The FITC-labeled liposomes (drug free) were first diluted 400-fold in the media, and then one drop was placed on a slide for observation with a fluorescence inverted microscope (DMI 4000B, Leica, Germany). Ten second movies (frame rate: 37 fps) were captured at a temporal resolution of 32.6 ms using LAS 4.5 software (Leica). The tracking process of the nanoparticles was analyzed using ImageJ software (NIH, Bethesda, MA, USA)32 (link). Three independent measurements were conducted for each sample and 100 particles were analyzed in each test. The accumulative mean square displacement (MSD) and effective diffusivities (Deff) were calculated using Eqs. (5), (6), respectively: MSD=[x(t+τ)x(t)]2+[y(t+τ)y(t)]2 Deff=MSD/(4τ) where x and y are the coordinates of the liposomes in PEO hydrogels and τ is the time scale33 (link),34 (link).
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8

Larval Growth Evaluation Protocol

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The initial sampling was conducted after the acclimatization period (22 dph), prior to the beginning of the experiment. From each tank, 25 larvae were randomly collected and sacrificed with ice-cold water. A subsample of twenty larvae was then measured for total length under a microscope (Leica DMC2900, Wetzlar, Germany) using the LAS 4.5 software (Leica, Wetzlar, Germany). After measurements, a subsample of 15 larvae was placed in 3 groups of 5 larvae on cover glasses previously weighed to determine the wet weight. The glasses were then placed in a laboratory oven at 60 °C for 48 h to determine the dry weight. In addition, 5 larvae were also placed in RNAlater solution, stored at 4 °C for one week, and then frozen at −80 °C until gene expression analyses.
The same procedure was also used to sample larvae at 29 dph (intermediate sampling) and 36 dph (final sampling). At the latter sampling point, a pool of 17 larvae from each tank was also collected for biochemical analyses, frozen at −80 °C, and subsequently lyophilized until analyses could be conducted.
Furthermore, 3 different samples of enriched rotifers and Artemia sp. metanauplii were collected, frozen at −80 °C, and lyophilized until biochemical analyses. Samples of the inert diets were also stored at −80 °C until analyses. The biochemical and fatty acid composition of the diets is presented in Table 1.
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9

Immunofluorescence Imaging of Tumor Cells

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For immunofluorescence, tumor cells were obtained as described in Rowald et al. (2016) (link). Cells were cultured on coverslips coated with Collagen I (Cultrex, 344.020-01). After 24 h, cells were fixed with 4% PFA for 10 min at room temperature or with a methanol/acetone mix for 10 min at −20°C and permeabilized with 0.2% Triton X-100 PBS for 10 min. Cells were treated with blocking buffer containing 5% donkey serum, 1% BSA, and 0.2% Triton X-100 in PBS for 1 h at RT. The following primary antibodies were used: rabbit anti-LC3 (1:100, Sigma, L7543), rabbit anti-Fibronectin (1:40, Abcam, ab23750), mouse anti-Ataxin1 (1:50, Santa Cruz, sc-365343), mouse anti-Myosin X (1:50, Santa Cruz, sc-166720), mouse anti-RhoA/RhoC (1:100, Thermo Scientific, 1B3-4A10), mouse anti-Vimentin (1:50, Sigma, V2258), and rabbit anti-AKT3 (1:800, Cell Signaling, E1Z3W). Donkey anti-mouse Alexa 488 and anti-rabbit Alexa 568 secondary antibodies were used (1:500, Invitrogen), and the DNA was stained with DAPI. Images were acquired with the Leica LAS 4.5 software on a Leica SP5 confocal microscope. Quantification of LC3 fluorescence intensity was performed with ImageJ software.
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10

Probing Mucus Dynamics: Microscopic Analysis

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We euthanized male Sprague-Dawley (SD) rats, and carefully isolated the small intestinal to collect the fresh rat mucus. And a SNE formulation (25 mg/ml in HBSS, 2 µL) was placed on the fresh mucus (200 µL), followed by a 30 min incubation at 37 °C. Movies were taken at a temporal resolution of 32.6 ms for 10 s using the LAS 4.5 software (Leica, Germany). The tracking resolution was about 10 nm, determined by gluing microspheres on microslides and tracking their apparent displacement by referring to a previous report [22] (link), [23] (link). The trajectories for n = 100 particles were analyzed using Image J, and three independent experiments were performed. Time averaged mean square displacement (MSD) and effective diffusivities (Deff) were calculated using the following equations:
MSDt=(xtx0)2+(yty0)2Deff=MSD/(4t)
(Where x and y represent the coordinates of the particle, and τ represent time scale or time lag).
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