Ms 222

One-year-old brown trout and rainbow trout, sampled at each site, were killed with an overdose MS-222 (tricaine mesylate, Sigma-Aldrich, St. Louis, USA). Blood samples were taken immediately from the caudal vein by a sterile syringe, transferred in lithium-heparinized reaction tubes (Co. Sarstedt, Germany), and 4 TIU aprotinin (C. Roth, Germany) per mL blood was added. Samples were centrifuged (4 °C, 10 min, 2500 rpm Eppendorf 5810R) on-site and plasma samples were snap-frozen in liquid nitrogen. Thereafter, plasma aliquots were stored at −80 °C until we determined VTG levels. After taking the blood samples, the length and weight of each fish were measured, gonads were removed for histological examinations and fixed in 2 % glutaraldehyde dissolved in 0.1 M cacodylic acid.
Larvae were killed with an overdose MS-222 (tricaine mesylate, Sigma-Aldrich, St. Louis, USA), and the region between head and pectoral fin from each individual was placed in Eppendorf tubes, snap-frozen, and stored at −80 °C.
All the following steps were undertaken on ice. Homogenates of juvenile trout were prepared by adding homogenization buffer (4 times the sample weight; PBS + 2 TIU aprotinin, C. Roth, Germany), mixing with a plastic pestle, centrifuging (10 min, 4 °C, 20,000×g Eppendorf 5810R) [17 (link)] and storing the supernatants at −80 °C.

Embryos were anesthetized with MS-222 (Sigma) and embedded in 0.8% agarose (Fisher) for imaging. Epifluorescence images were acquired with an Axiovert 200 M (Zeiss) and an Orca-ER camera (Hamamatsu). Bright field images were acquired with an MZ16F L3 (Leica) and Axio Cam HR (Zeiss). Confocal images were acquired with an LSM 880 (Zeiss). Images were analyzed in FIJI^{87 (link),88 (link)}. For time-lapse microscopy of Tg(col9a2:mCherry; col8a1a:GFP) embryos, embryos were anesthetized in holtfreter’s buffer (59 mM NaCl, 0.88 mM CaCl₂·2H₂0, 0.67 mM KCl, 5 mM HEPES) containing 600 µM MS-222 (Sigma) and embedded in 1% low-melt agarose (NuSieve GTG) containing MS-222 on glass-bottomed microwell dishes (MatTek 35 mm). Imaging was performed using a Nikon W1 spinning disc confocal microscope. After acquisition, image sequences were bleach corrected using histogram normalization⁸⁹ , and channels merged in FIJI.

Larval development (biometry), mortality, and gene expression of selected genes, corresponding to specific molecular mechanisms were followed from hatch and throughout the endogenous feeding stage (2, 4, 6, 8, 10, and 12 dph). All endogenously feeding larvae of European eel were anesthetized using tricaine methanesulfonate (MS-222; Sigma-Aldrich, Missouri, USA) prior to digital imaging and euthanized post-sampling by using an MS-222 overdose [13 (link)]. All images were taken using a digital camera (Digital Sight DS-Fi2, Nikon Corporation, Japan) attached to a zoom stereomicroscope (SMZ1270i, Nikon Corporation, Japan), while NIS-Elements D analysis software (Version 3.2) was used to analyze the larval images (Nikon Corporation, Japan).

Animal experiments were carried out in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care International (http://www.aaalac.org/index.cfm), and protocols were approved by the Institutional Animal Care and Use Committees (IACUC) of Tongji University.
For limb amputation, animals were anesthetized in 1/10 MMR containing 0.02% MS-222 (from stock of 0.5% MS-222, Sigma-Aldrich, United States, pH 7.5 buffered with Tris-Cl), NF stage 52–53 tadpole hindlimbs were amputated with a surgical scissor at the level of presumptive knee, and froglet forelimbs were amputated at mid-ulna/radius (Zhang et al., 2018a (link)). For tail amputation, NF stage 49–51 tadpoles anesthetized were amputated with a surgical blade perpendicular to the notochord, removing 40% of the tail (Lin and Slack, 2008 (link)).

Eels were either killed with an overdose of neutralized tricaine methanosulfonate (MS-222; Sigma-Aldrich, St. Luis, MO, USA), or anesthetized with MS-222 and subsequently decerebrated and spinally pithed. The swimbladder was dissected, freed from connective tissue to reveal the actual gas gland tissue, cleaned from Anguillicola crassus specimen if necessary, immediately shock frozen in liquid nitrogen, and stored at -80°C until further use. Infected swimbladders contained between 5 and 30 parasites, and the swimbladder wall was markedly thickened and nontransparent as stated previously [30 (link)]. Tissue sampling was performed in compliance with the Austrian law, the guidelines of the Austrian Federal Minister for Education, Arts, and Culture, and also the Dutch and German law. The tissue sampling procedure was approved by the Tierversuchskommission of the University of Innsbruck.

To observe the morphology, we treated embryos with 0.04 mg/ml MS-222 (Sigma) at room temperature for 5 min. Embryonic pictures were taken by an Olympus MVX10 microscope (Japan), and the photos were improved by Photoshop. The transgenic zebrafish lines Tg(hb9:eGFP), atrn^nju70 and Tg(olig2:dsRed), and atrn^nju70 were to confirm the development of motor neurons and oligodendrocytes. All confocal images were acquired using a Zeiss LSM880 confocal microscope. Then when measuring the weight and length of zebrafish, we treated them with 0.04 mg/ml MS-222 (Sigma), then blot the water, placed it in a Petri dish, measured it with an analytical balance, and recorded the data.

The fish were euthanized with Tricaine Methane Sulfonate (MS-222) to collect the spleen and thymus (Sigma-Aldrich; St. Louis, MO) at 200 mg/L. MS-222 was administered within 2 minutes of catching the fish to reduce stress from handling. Aseptic techniques were used for the removal of the spleen and thymus. The isolated spleen and thymus were kept in a 1x phosphate buffer solution (PBS) for the cell culture.