Hm 325 rotary microtome

At the time of collection, mice tissue samples (perfused lungs, dorsal skin, inguinal lymph nodes or tumors) were collected and fixed overnight in PFA 4% at 4 °C. Samples were then washed three times in PBS, processed in an STP 120 Spin Tissue Processor (ThermoFisher Scientific, MA, USA; cat# 813150) overnight and embedded in paraffin (Hounisen, DK; cat# 2270.6060) using a Microm EC 350 modular tissue embedding center (ThermoFisher Scientific, MA, USA). Unless otherwise indicated, sections of 4 µm were cut using an HM325 rotary microtome (ThermoFisher Scientific, MA, USA; cat# 902100), MX35 Premier blades (ThermoFisher Scientific, MA, USA; cat# 3051835) and ThermoScientific™ SuperFrost Plus™ Adhesion slides (ThermoFisher Scientific, MA, USA; cat# J1800AMNZ). Sections were deparaffinized with xylene and graded ethanol, and rehydrated. Sections were then stained according to various staining procedures.

Tumour samples were fixed overnight in 4% paraformaldehyde at 4 °C, dehydrated in graded ethanol baths, cleared with xylene, embedded in paraffin and cut into 4-µm-thick sections using the HM 325 Rotary Microtome (Thermo Fisher Scientific). These sections were mounted onto Superfrost plus slides (Epredia, J1800AMNZ) and allowed to dry for 2 days at room temperature. Haematoxylin and eosin staining was performed at the EPFL Histology Core Facility using the Ventana Discovery Ultra automated slide preparation system (Roche).

For pollen grains and pollen tube visualization in the stigma, 1 DAP carpels were fixed in FAA (5% glacial acetic acid, 3.7% formaldehyde, and 50% ethanol). Callose in the pollen was revealed by aniline blue staining, where callose fluorescence was detected at 520 nm, according to Longbottom et al.^{51 (link)}, in a Nikon microscope Eclipse 80i (Nikon, Japan) using NIS Element software.
For carpel/berry mesocarp growth observations, samples collected at 2, 6, 10, and 12 DAP were fixed in FAA solution. Afterward, fixed samples were vacuum treated and passed through increasing ethanol series for tissue dehydration, then paraffin-embedded and cut into 6–8 μm transverse sections in an HM 325 Rotary Microtome (Thermo Scientific™, Thermo Fisher Scientific, USA). Sections were stained with 0.05% toluidine blue in sodium acetate buffer. Images were obtained using a light microscope (Olympus BX51). Cell number per transverse section, cell diameter, and mesocarp width were obtained through tissue observation using the open-source software ImageJ®⁵² . For mesocarp width, cell number, and diameter, a row of cells from inner epidermis to outer epidermis was measured (including inner plus outer mesocarp). These parameters were determined in eight biological and two technical replicates per time point. Technical replicates were averaged.

The cell pellets were fixed in 20 volumes of methanol (A601617, BBI) for 10 min at room temperature, washed with PBS, mixed with 4% melted agarose (75510-019, Invitrogen) and placed in a mold. After cooling for 5 minutes, agarose blocks were placed in plastic cassettes and underwent standard formalin fixation, dehydration and paraffin block preparation.
The paraffin blocks containing control cell lines were sections with HM325 rotary microtome (902100, Thermo) to obtain triplicates sections with different thickness.

The liver was collected after anesthesia by isoflurane, then washed with physiological saline and fixed in 4% formaldehyde solution for 72 h. Then, the fixed liver was embedded in paraffin and prepared for sliding with an HM 325 Rotary Microtome (Thermo Scientific, Waltham, MA, USA) for fat granules observation. The images of sections were captured using an OLYMPUS BX43 Light Microscope (OLYMPUS, Japan) [33 (link)].

Liver hematoxylin-eosin staining was evaluated in three rats per group. Rats were anesthetized, and each section of the liver was swiftly extracted and submerged in 10% formalin for 2 h. Afterward, the liver was rinsed three times with PBS. After dehydration and embedding in paraffin, the liver tissues were sliced into 6-µm-thick sections using a Thermo Fisher Scientific HM-325 rotary microtome (Waltham, MA, USA) for hematoxylin-eosin staining. The nuclei were counterstained with hematoxylin and differentiated with eosin in the cytoplasm. Using an AxioLab A1 light microscope (Zeiss, Jena, Germany), high-magnification images were captured. A minimum of five fields were evaluated for each slide. A numerical scoring for assessing histological activity was carried out in accord with Knodell et al. [34 (link)].