Eg1140h

Moribund fish showing clinical signs (~10) were anesthetized with Clove oil (at 50 µl per liter of water) and used for collection of tissue. The post-mortem examination was done to observe and record gross lesions in internal organs of fish. Tissue samples from liver were collected and fixed in neutral buffered formalin (NBF) (10%). The fixed tissues were washed and cut into 1- to 2-mm small pieces. Then, the samples were dehydrated in ethanol with different concentrations followed by treatment with the clearing agent xylene. The cleared tissue was embedded into paraffin using an impregnation technique (Leica EG 1140H, Germany). Sectioning of the paraffin-embedded tissue was done using a microtome, maintaining a thickness of 5 μm, followed by staining with hematoxylin and eosin (35 (link)). Afterwards, the sections were observed under a light microscope for cellular and pathological changes.

Adult mice were subjected to the surgical procedure described above, in order to transplant hMSCs into the deep cerebellar nuclei of the cerebellum. Five different post-transplant timepoints were determined to confirm hMSC survival in the mouse brain: 48 h, 1 week, 4 weeks, 6 weeks, and 8 weeks post-transplantation of hMSCs. At each timepoint, mice were deeply anaesthetized and transcardially perfused with PBS, followed by 4% PFA in PBS. The brains were post-fixed overnight in fixative solution and embedded in paraffin (EG1140H, Leica, Germany); 4 μm thick paraffin sections were processed via immunohistochemistry for human nuclear antigen (HNA; MAB1281, 1:150, Merck Millipore, Lisbon, Portugal), and then developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate (D5905, Sigma, Germany). Slice microphotographs (total 3–4 sections) were acquired using DPController software (Olympus, Spain) and a camera (Olympus DP70, Spain) attached to a motorized microscope (4E14135, Olympus BX61, Spain) using a 4 × 0.16 numerical aperture objective.

Both electronic endoscopy and binocular optical microscope were obtained from Olympus (Nagano, Japan; product model of electronic endoscopy: GIF-H290, GIF-Q260, and GID-XQ260; product model of binocular optical microscope: BX50F-3). Both tissue dehydrator and paraffin embedding machine were obtained from LEICA (Germany; product model of tissue dehydrator: TP1020; product model of paraffin embedding machine: EG1140H). A Rotary paraffin slicer was obtained from SHANDON (UK; product model: HANDON-AS325). A biological tissue sheet roasting machine was obtained from research institute of Yaguang medical electronic technology (Hubei, China; product model: YT-6). An electric constant temperature drying oven was obtained from Yuejin medical apparatus factory (Shanghai, China; product model: 101-1-BS). Polyclonal antibody of COX-2 was purchased from Abcam (Shanghai, China; batch no.GR287158). An Elivision plus kit was obtained from MAXIN (Fujian, China; batch no.1505074928). Rapid urease test (RUT) paper was purchased from Kedi Technology (Zhuhai, China; batch no.180102). A diaminobenzidine (DAB) color developing agent was purchased from Boster bioengineering co. LTD (Hubei, China; batch no.10109A32).

Tissue samples from the brain, lungs, liver, heart, spleen, thymus, kidneys, adrenal glands and sex organs were routinely processed for histology and fixed in buffered formalin 10% (Panreac©, Barcelona, Spain, stabilized with methanol at pH 7) for 24 h at room temperature. Samples were then embedded in synthetic paraffin (Casa Alvarez, Madrid, Spain) with a melting point of 56 °C, using an automatic tissue processor (ASP300, Leica^®, Wetzlar, Germany) with a program of automatic transmissions alcohols increased histological grading. Blocks were performed in a block forming unit (console Leica© EG1140H and cold plate Leica© EG1130), and 4 micron thick sections were obtained from rotation microtome Leica© brand, model RM 2155. Sections were deparaffinized in xylene and hydrated in alcohol and water. Conventional staining method, hematoxylin and eosin, was used by means of Leica© auto stainer SP4040. Then, dehydrated first ascending alcohol series and xylene were used, and finally, mounted with DPX (Nustain©, Nottingham, UK). The slides were then observed under a light microscope.

The lateral skin samples from collected fishes were cut into 0.8 cm × 1.0 cm × 0.2 cm pieces and fixed with 10% neutral formaldehyde for 10‒24 hr, washed, and dehydrated through a graded series of ethanol (50%, 70%, 80%, 95%, and 100%). Paraffin embedding was carried out using a paraffin embedding station (Leica EG1140H; Leica Microsystems). The paraffin blocks were sliced into 6‐μm‐thick sections (Leica RM2016; Leica Microsystems), dried at 38°C for 7‒24 hr in the oven, and stained with haematoxylin–eosin. Sections were imaged using an optical microscope (OLYMPUS BX 51; Olympus).