Histocore autocut

Tissue samples for histology were collected and processed in half of the cases (n = 19). They were fixed in 10% formalin for 24 h, dehydrated, and included in paraffin using the Donatello Diapath automatic tissue processor (Martinengo, Bergamo, Italy), sliced (HistoCore Autocut, Leica BioSystems microtome) with 2 μm thickness, and stained with hematoxylin and eosin using the automated Dako CoverStainer (Santa Clara, CA, USA). Each section was documented at 20× and 40× magnification, by using the Olympus BX51 microscope and connected with an Olympus DP70 digital camera and AnalySIS 5.0 imaging system software (Olympus, Tokyo, Japan). Analyses were adapted from the method of Kleiner et al. [21 (link)] to evaluate and score (yes/no, 1/0) vessel dilatation, fibrosis, portal and lobular inflammation (also graded by foci number at 20× magnification, 1 = one focus, 2 = two-four foci, 3 = >four foci), or ballooning degeneration, and micro/macrovesicular steatosis (also graded as the percentage of the affected cells). The diagnosis of non-alcoholic steatohepatitis was defined as the sum of grades ≥4 (sum score: range 4–7, including steatosis, lobular inflammation, and ballooning), and categorically (non-alcoholic steatohepatitis, NASH yes/no).

The tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin and cut into 4 µm sections with HistoCore Autocut (Leica Biosystems, MA, USA). Then, the deparaffinized and rehydrated sections were stained with the primary antibodies anti-CD3ε rabbit mAb (Cell Signaling Technology, MA, USA) at 1:200 dilution, anti-CD8α rabbit mAb at 1:200 dilution (Cell Signaling Technology), and Brilliant Violet 605 anti-mouse CD69 at 1:100 dilution (BioLegend) and labeled with a TSA-Rab multiplex immunofluorescence staining kit (Panovue, Beijing, China) according to the manufacturer’s instructions. The cell nuclei of the sections were stained with DAPI before being visualized by an LSM880.

For each group, the whole hepatopancreas from 5 shrimps were collected for histological analysis. After being fixed in 4% paraformaldehyde for 24 h, the hepatopancreas tissues were dehydrated using a gradient solution of methanol (25%, 50%, and 75%) with each step lasting 30 min and embedded in paraffin using embedding equipment (Leica Biosystems HistoCore Arcadia, Leica Biosystems, Wetzlar, Germany). The sections were continuously sliced with a fully automated rotary microtome HistoCore AUTOCUT (Leica Biosystems, Wetzlar, Germany) to 4~5 μm thicknesses, spread in a water bath at 40 °C, mounted on glass slides (three to four serial sections per slide), and dried at 37 °C overnight. The wax was removed from the hepatopancreas tissue sections using xylene and ethanol, rehydrated in a decreasing ethanol gradient (100%, 95%, 80%, 70%, 50%, and 30%; 2 min for each step), and stained with hematoxylin–eosin (H&E) (Njjcbio, Nanjing, China). All the stained slides were dehydrated in ethanol and xylene (Macklin, Shanghai, China) for 5 min each, sealed with neutral gum (Solarbio, Beijing, China), and then observed under a BX43 manual light microscope (Olympus, Tokyo, Japan).

Testicles and prostates were fixed in 4% neutral buffered paraformaldehyde solution for 24 h, followed by dehydration in increasing concentrations of ethanol and xylene and then embedded in Histowax (Histolab Product AB, Askim, Sweden). A rotary microtome HistoCore AUTOCUT (Leica Biosystems, Wetzlar, Germany) was used to obtain 5 μm thick slices. Hematoxylin-eosin (H&E) staining [45 ] was used for testicular and prostate tissue visualisation. Microscope slides were examined using light microscopy Leica DM LS2 (Leica Microsystems, Wetzlar, Germany) while original photographs were made using Canon Power Shot S70 camera (Canon U.S.A. Inc., Huntington, NY, USA).

Femurs were collected in four mice (two hypomorphic, two control mice). They were fixed in 10% formalin for 48 h and then in 70% ethanol for 30 minutes. Samples were rinsed in distilled water and incubated with decalcifying solution for two hours (DiaPath S.p.A., Microdec, EDTA-Based, Ref. D0053, Martinengo, Bergamo, Italy). The decalcification process was ended when the bone was easily penetrated by a needle. Then, femoral bone marrow was processed and included in paraffin using the Donatello Diapath automatic tissue processor (Martinengo, Bergamo, Italy), sliced (HistoCore Autocut, Leica BioSystems microtome) with thickness of 2 μm, and stained with hematoxylin and eosin using the automated Dako CoverStainer (Santa Clara, CA, United States). Each section was documented at 10×, 20× and 40× magnification, by using the Olympus BX51 microscope and connected with an Olympus DP70 digital camera and AnalySIS 5.0 imaging system software (Olympus, Tokyo, Japan).

The fixed flowers were washed twice, then stained with hematoxylin for four to 6 days (depended on the degree of development of the flower) at 45–50 °C. The fixed seeds in the different developmental stages were taken out from fruits, and soaked in sodium hydroxide solution or under running water for a period of time (adjusted according to the developmental degree of seeds), then washed twice and stained like the flowers. They were dehydrated in an ethanol gradient of 70 to 100% ethanol, transparentized in xylene, dipped in paraffin, embedded in paraffin and freeze it finally. Embedded materials were sectioned at 6-8 μm thickness using a slicer (HistoCore AUTOCUT; Leica, Germany) and the slices were treated with xylene. In the end, it was sealed with neutral balsam and the slides were observed and photographed using a light microscope equipped with camera.

H&E staining and IHC were performed as previously described [36 (link)]. The brain samples were embedded in paraffin blocks, and the sections were prepared by HistoCore AutoCut (Leica, Deerfield, IL, USA). Next, the sections were cut into 4 μm sections and stained with H&E, following standard procedures. For IHC, sections were treated with 3% hydrogen peroxide/methanol and then with 0.25% pepsin to retrieve antigens. Next, samples were incubated in blocking solution (Dako, Carpinteria, CA, USA), after which they were incubated at 4 °C overnight with the specific primary antibodies diluted in the antibody diluent (Dako). The sections were subsequently washed with tris buffered saline with 0.1% Tween 20 and then incubated with polymer-horseradish peroxidase-conjugated secondary antibody (Dako). A 3,3′-diaminobenzidine substrate chromogen system (Dako) was utilized to detect antibody binding. Stained sections were observed under an Olympus IX71 inverted microscope (Olympus Optical, Tokyo, Japan).