Cryotome fse

For histological studies fresh, frozen and fixed materials of leaflets of R. psudoacacia were used. During studies, 4 types of samples were used: control blade of leaflet without gall and gall at three studied stages (young, matured and senescence. Each morphological stages of gall formation have been tested at least on 10 different leaves. Material for histochemistry analysis was fixed chemically in FAA (formaldehyde, glacial acetic acid, ethyl alcohol) for 24 hours, initially under reduced pressure, at room temperature. The material after fixation was dehydrated in 50 and 70% ethanol [40 ].
The samples were cut trans-versely with a sharp razor blade into smaller segments and then into 10 μm sections on cryostat (Cryotome FSE, Thermo Scientific). At the same time handmade scraps were done by razor blade and viewed in water. Histological sections were stained with: Ehrlich haematoxylin, Lugol’s solution (starch detection), and Sudan III (lipid detection) [40 , 41 ]. Larger leaf fragments with whole galls were stained with Trypane blue, and phloroglucinol with hydrochloric acid (lignin detection). After staining, the slides were mounted on microscope slides in 50% glycerol. The anatomical observations were performed using an Olympus microscope (BX53 and BX61 with fluorescent unit) and Olympus SC30. Micrographs were taken using CellSense Standard program.

After the fattening period animals from each group were slaughtered, and samples of the lungs, liver, kidneys, and jejunum were taken. These samples were fixed for 24 h in 10% neutral buffered formalin and absolute alcohol, and then passed through increasing concentrations of alcohol solutions and xylene in a tissue processor (Leica TP-1050) and embedded in paraffin blocks. Histological Sects. 4 µm thick, prepared using a sledge microtome (Leica SR-200) and stained with haematoxylin and eosin, were used for morphological analysis under a light microscope. For the liver samples, histochemical staining was additionally performed for the presence of neutral fats. For these analyses, the sections were fixed in 10% neutral buffered formalin, sliced in a freezing microtome (Cryotome FSE, Thermo Scientific), and stained with Sudan IV according to Daddi [17 ]. For visualization of glycogen, microscope sections of the liver were fixed in absolute alcohol and stained by the PAS method according to McManus [18 ].

All endometrial tissues were immediately separated from the surgical specimen, embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) in a Tissue-Tek Cryomold (Sakura Finetek), frozen in liquid nitrogen, and stored at −80 °C. We cut 12-µm-thick serial frozen sections with a Cryotome FSE (Thermo Fisher Scientific) and mounted them on PEN-Membrane Slides (Leica).
For laser microdissection, the cryosections were fixed with 100% methanol for 3 min and then stained with toluidine blue for 30 s. Before laser microdissection, all images of cryosections were stored using the Specimen Overview function of the LMD7 laser microdissection microscope (Leica) to assess glandular continuity. We performed laser microdissection using LMD7 (Leica), distinguishing the vertical and horizontal glands, which often branched from or connected with each other (Fig. 7). The isolated epithelial tissues of each gland were collected in the caps of 0.2 ml PCR tubes (Axygen). The median number of frozen sections for sampling each gland was 13 sections (range: 8-18).

Mouse brains were harvested after formalin perfusion and cryopreserved using Optimal Cutting Temperature compound (Fisher Healthcare). Sections were cut 5-μm thick using Thermo Scientific Cryotome FSE cryostats. Tumors were confirmed using hematoxylin & eosin (H&E) staining, Ki67 (D3B5) (Cell Signaling Technology, #12202), H3.3K27M (D3B5T) (Cell Signaling Technology, #74829), and luciferase (Abcam, #ab185924) staining. Neurotoxicity was assessed via H&E and immunohistochemistry for glial fibrillary acidic protein (GFAP) (Agilent, #Z033429-2), Ionized calcium binding adaptor molecule 1 (Iba1) (Abcam, #ab5076), and cluster of differentiation 3 (CD3) (Abcam, # ab16669). Staining and immunohistochemistry were performed by the Weill Cornell Pathology Core. Slides were reviewed by D.J.P., a board-certified neuropathologist, for neurohistopathological changes observed in the treatment group against vehicle-treated animals.

To prepare frozen sections for immunofluorescence analysis, testes were fixed in 10 ml 4% paraformaldehyde (PFA) at 4 °C overnight. The next day, tissues were dehydrated with 15% sucrose in 1x PBS until totally sunk to the bottom of the tube, transferred to 30% sucrose for further dehydration at 4 °C overnight. And then, embedded in OCT (Fisher Scientific, 14-373-65) and cut into 5 µm in cryostat (Thermo Scientific Cryotome FSE). Slices were stored at −80 °C. For immunofluorescence analysis, the slices were treated as follows: air dried at room temperature, washed twice with PBS, permeabilized with 0.5% Triton X-100 in PBS at room temperature for 10 min. After washing three times with PBS, samples were blocked with 5% serum in TBS-T (TBS, 20 mM Tris, 150 mM NaCl, pH 7.6, and 0.1% Tween20) at room temperature for 1 h, incubated with primary antibodies in suitable concentration at 4 °C overnight, and then conjugated with secondary antibody and DAPI at 37 °C for 1 h. Chromosome spreads of prophase I spermatocytes were conducted as described previously^{45 (link)}. All fluorescence images were taken and analyzed by Carl Zeiss LSM800 confocal microscope or Image-Pro Plus 6.0. All statistical results were performed with Graph Pad Prism 6.

Standard protocols and data analysis methods for non-invasive PET imaging of small animals have been established in our group [20 (link)]. Healthy male Wistar rats (Japan SLC, Inc., Shizuoka, Japan) each weighing 200–250 g were utilized. The rats were anaesthetized with 2 % isoflurane. Ten to 20 MBq of [¹⁸F]AF78 were administered via tail vein injection. Ten minutes after radiotracer administration, the animals were euthanized. Hearts, livers and blood were obtained for ex vivo analysis with autoradiography (Typhoon FLA 7000) and tissue counts with a γ-counter (1480 WIZARD™ 3″). Following weight and decay correction of tissue counts, the heart-to-blood (H/B) and heart-to-liver (H/L) count ratios were calculated. For autoradiography, the rats under anaesthesia were first injected via tail vein either with or without the NET blocker phenoxybenzamine (50 mg/kg). After 10 min, the radiotracer (10–20 MBq) was administered. The hearts were harvested 10 min later, frozen, and cut into 20 μm short axis slices using a cryostat (Cryotome FSE, Thermo Fisher Scientific), which were then immediately exposed to the autoradiography plates (Fuji SR-type image plate, Fujifilm Corporation, Tokyo, Japan) for 18 h to obtain the radiotracer distribution. The images were obtained using a digital autoradiographic system (Typhoon FLA 7000).

Hooves were taken from freshly slaughtered cattle 2–3 years of age, stripped of adhering cartilaginous, connective tissue, and kept in distilled water for 72 h [18 (link)]). Sections, approximately 60 µm thick, were taken from the bottom of the hoof using a cryotome (Thermo Scientific Cryotome FSE). Prepared hoof membranes were kept at −20 °C and moved to room temperature 5 h before use in penetration studies.
Nail clippings were obtained from healthy human volunteers (male and female, aged 25–40 years) using nail clippers. Nail clippings were washed with phosphate buffer (pH 7.4) and wiped with filter paper.