Paraffin slicer

A group of mice was sacrificed on Day 6 and their skin samples were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and stored at RT until used. Paraffin sections (8 μm) were cut using a paraffin slicer (Leica, Solmas, Germany) and stained with H&E (Beyotimes, Shanghai, China). Images of H&E staining were acquired using an eclipse upright optical microscope digital camera (Nikon, Tokyo, Japan). Two random positions from each mouse were selected to measure the epidermal thickness by Image J software (version 1.8.0, Maryland). Baker’s scores (29 (link), 29 (link)) were used to analyze the pathological severity of the skin. CD11c positive cells were stained with biotin-rat anti-mouse CD11c antibody (1:100, Biolegend, California) for 1 h, followed by streptavidin-HRP antibody (1:800, Yeasen, Shanghai, China) incubation for 40 min. Sections were developed with DAB (Vector Laboratories, California) and counter-stained with hematoxylin (Phygene, Shanghai, China) before visualization under the microscope (Nikon). CD11c positive cells (30 (link)) were counted in 5 microscopic fields under 200X magnification and expressed as cells/field, and mean ± SEM.

The specific primers were designed with Primer 5.0 (Table 1), and the purified PCR products were used as in vitro transcription templates to obtain labeled probes using the T7 High Efficiency Transcription kit (TransGen Biotech, Beijing, China) and DIG RNA Labeling Mix (Roche, Germany), and the probes were purified.
The mantle tissues of the mussels were fixed in 4% paraformaldehyde for 24 h at 4 °C and stored in 70% ethanol (prepared DEPC water). The tissues were gradient-dehydrated, embedded, and sectioned with a paraffin slicer (Leica, Heidelberg, Germany) at a thickness of 4 μm. Then, in situ hybridization was performed according to the instructions of the DIG nucleic acid detection kit (SP6/T7; Roche, Germany), treated with DAB chromogen for light avoidance, and the hybridization signal was observed and photographed using a Leica DM 2500 microscope (Leica, Heidelberg, Germany).

On days three and six, hepatopancreas samples were collected for histopathological analysis. The samples were excised and fixed in 40 g L⁻¹ paraformaldehyde at 4 °C overnight [51 (link)]. The samples were then dehydrated in ethanol, cleared in dimethyl benzene, and embedded in paraffin. The paraffin-embedded sections (5 μm) were sliced with a paraffin slicer (Leica, Wetzlar, Germany) and stained with hematoxylin and eosin (H&E). Finally, the sections were observed under a light microscope to identify histological changes (Leica, Wetzlar, Germany) [52 (link)].

Low-temperature high-speed centrifuge (MIKRO 220, Schwartzwald, Germany), paraffin slicer (Leica, Frankfurt, Germany), slicer (Leica), optical microscope (Leica), vertical electrophoresis tank (Bio-Rad, Hercules, CA, USA), transfer electrophoresis tank (Bio-Rad), and fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA) were used for the experiments.

The anterior midgut was dissected and fixed in 4% paraformaldehyde overnight at 4 °C, dehydrated in a graded ethanol series, embedded in paraffin, and sectioned at 5 μm thickness by using a paraffin slicer (Leica, Wetzlar, Germany). The slices were stained with hematoxylin and eosin and examined by light microscopy (Olympus, Tokyo, Japan).

The following instruments were used: electronic balance (Shanghai Jingtian Balance Factory); small electric drill (Dremel, USA); optical microscope (Olympus BX51, Japan); paraffin slicer (Leica, Germany); and video Vickers hardness tester (mhv-10z, Shanghai Binocular).