Processing of undecalcified bone specimens and cancellous bone histomorphometry were performed as described25 (link),70 (link). Bone specimens (femur, tibia, and vertebra) were dehydrated and embedded in methylmethacrylate. Sections (5 μm in thickness) were prepared using a microtome (#RM2235, Leica) microtome and were stained by the von Kossa/nuclear fast red method. For hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase (TRAP) staining, bone specimens were decalcified in 10% EDTA for 2 weeks at room temperature. After serial dehydration in a series of ethanol (70–100%), samples were embedded in paraffin. Sections (5 μm in thickness) were processed by a microtome (#RM2235, Leica) ready for staining. Histomorphometric measurements were performed using OsteoMeasure software (OsteoMetrics, Decatur, GA)71 (link).
Rm2235
Manufactured by Leica
Sourced in Germany, United States, China, Italy, United Kingdom, Japan, Brazil
The RM2235 is a rotary microtome designed for sectioning paraffin-embedded tissue samples. It features a vertical specimen feed and a high-quality steel knife or disposable blade holder. The RM2235 allows users to obtain consistent, high-quality tissue sections for further analysis and investigation.
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Lab products found in correlation
Tp1020, by Leica (12 mentions)
Asp300, by Leica (11 mentions)
Dm1000, by Leica (11 mentions)
Dm2500, by Leica (10 mentions)
Lsm 880, by Zeiss (8 mentions)
Eg1150h, by Leica (8 mentions)
Image pro, by Media Cybernetics (8 mentions)
Axioplan light microscope, by Zeiss (7 mentions)
Axiocam mrc5, by Zeiss (7 mentions)
Dm3000, by Leica (7 mentions)
Hematoxylin, by Merck Group (6 mentions)
Eg1150, by Leica (6 mentions)
Ds fi1, by Nikon (6 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (5 mentions)
Novared, by Vector Laboratories (5 mentions)
Citra plus solution, by BioGenex (5 mentions)
Eclipse e200, by Nikon (5 mentions)
Neutral buffered formalin, by Bio-Optica (5 mentions)
Image pro plus 6, by Media Cybernetics (5 mentions)
Eclipse 80i, by Nikon (4 mentions)
606 protocols using rm2235
1
Undecalcified Bone Histomorphometry
2
Microscopic Analysis of Plant Tissues
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All of the samples of experiment II at 1, 5, 9, 25, and 30 DAG were placed in FAA for 1 d and then were dehydrated in an ethanol series (50%, 60%, 70%, 80%, and 95%), with 60 min at each dehydration step. In the last step, samples were placed in 100% ethanol overnight. After decoloration with dimethylbenzene, the samples were embedded in paraffin. The samples were sectioned vertically to 10 μm using a rotary microtome (Leica RM2235, Germany, Wetzlar), and were dewaxed, rehydrated, cleaned, stained with toluidine blue, counterstained with safranin, and then fixed with neutral balata. The sections were examined and photographed using an optical microscope (Leica RM2235).
3
Intestinal Morphometry and pH Profiling
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The intestinal samples (n = 12/treatment) were dehydrated with gradient concentrations of ethanol, transparentized with xylene, embedded in paraffin and sectioned (3 μm thickness per section) using a sliding microtome (Leica RM2235, Leica Microsystems, Wetzlar, Germany), then stained with hematoxylin and eosin solution. The length of the villus and the depth of crypt of each segment of the small intestines were analyzed using Image-Pro Plus 6.0 software. The contents of the duodenum, jejunum, ileum and cecum were diluted (1:50) using ultrapure water. The mixtures were homogenously mixed using a vortex mixer (XH-C, Mai Xingkang Co., Ltd, Taizhou, China) then the pH values were measured using a pH meter (PHSJ-4F, Rex, INESA Scientific Instrument Co., Ltd, China).
4
GFP Immunostaining of Root Sections
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Immunostaining was performed according to the methods in ref. 68 (link). Briefly, transgenic hairy-root segments (10 mm from root tips) were kept in fixative solution [4% (w/v) paraformaldehyde, 60 mmol/L sucrose, and 50 mmol/L sodium cacodylate (pH 7.4)] for 2 h prior to slicing 100-μm-thick sections with a microtome (LEICA RM2235, Leica Microsystems GmbH, Wetzlar, Germany). Sections were incubated with 0.3% (v/v) Triton X-100 for 2 h, followed by the anti-GFP (1:1000; Thermo Fisher Scientific, Somerset, NJ, USA) primary antibody incubation overnight and the secondary antibodies (Alexa Fluor 555 goat anti-rabbit IgG; 1:2000; Molecular Probes, Eugene, OR, USA) for 2 h. Fluorescence was observed with a confocal scanning microscope (LSM880, Carl Zeiss, Oberkochen, Germany).
5
Histological Evaluation of Intestinal Inflammation
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Collected samples were fixed in 4% paraformaldehyde at 4°C overnight, dehydrated, and embedded in paraffin and cut into sections using a Leica RM2235 microtome ( thick; Leica Microsystems) and stained with hematoxylin and eosin for light microscopy examination. The slides were reviewed in a blinded fashion by a pathologist and were assigned a histological score for intestinal inflammation, ranging from 0–4 (Table 1 ).
6
Quantification of Medaka Larval Adipocytes
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After the feeding tests, the medaka larvae were collected, sacrificed by immersion into ice chilled water, and fixed with 4% paraformaldehyde solution in PBS. According to manufacturer’s instruction, the medaka larvae were embedded in plastic resins (Technovit 8100; Heraeus Kulzer, Hanau, Germany). The tissue blocks were cut into serial thin sections (thickness, 5 μm) with a rotary microtome LEICA RM2235 (Leica Microsystems, Wetzlar, Germany) and placed onto glass slides. The sections were stained with hematoxylin and eosin (H&E), and then, the number and area of the adipocytes and area of the tissues on the randomly selected sections (three sections per fish) were measured using ImageJ 1.43r. Data were expressed in terms of the median and interquartile ranges. Statistical analyses were performed using the Mann–Whitney U test or Steel–Dwass’ test for multiple comparisons.
7
Histomorphometry of Longissimus Dorsi Muscle
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Histomorphometric properties of 30 LD muscles (n = 10 LD muscles/diet) were determined. Each 1 × 1 cm piece of muscle from the same location of the LD muscle’s middle section was removed, and it was fixed in 10% neutral buffered formalin. Tissue sections of 4 mm thickness were cut using a rotary microtome Leica RM 2235 (Leica Microsystems, Nussloch, Germany) and stained with hematoxylin and eosin, followed by the standard paraffin embedding technique. Using an Olympus BX63 microscope (Olympus Corp., Tokyo, Japan), an Olympus DP72 digital camera (Olympus Corp., Tokyo, Japan), and a computer running the Image-Pro Plus application system for Windows, version 7.0 (Media Cybernetics, Inc., Bethesda, MD, USA, 2009), prepared Longissimus dorsi muscle histologic preparations were analyzed. LD muscle fibers’ cross-sectional areas (fiber length; 150 fibers were measured in each sample in three fields of view), as determined morphometrically, are expressed in micrometers squared (µm2).
8
Quantification of Stria Vascularis Thickness
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hematoxylin staining was used to visualize nucleic acids of the cells in the cochlea and measure stria vascularis thickness [99 (link),101 (link)]. Harvested tissue samples were placed in 4% paraformaldehyde in PBS for 24 h, at 4 °C, decalcified in 10% EDTA for 1 week at room temperature, embedded in paraffin, sectioned on a mechanical implant microtome (Leica RM2235, Leica Microsystems, Wetzlar, Germany) at a thickness of 4 μm, and stained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA). The stained tissue sections were photographed using a slide scanner (Panoramic MIDI version 1.23, 3DHISTECH, Ltd., Budapest, Hungary) and the numbers of hematoxylin-positive cells were quantified.
9
Histomorphometric Analysis of Implant Vascularization
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The implants extracted 2 and 8 wk after implantations that were placed in 10% neutral buffered formalin were decalcified in 10% EDTA solution (pH 7.4), dehydrated in ascending concentrations of ethanol and embedded in paraffin. Paraffin tissue blocks were sectioned (4 μm) on a microtome Leica RM2235 (Leica Microsystems, Solms, Germany). Obtained tissue sections were deparaffinized with xylene and stained with hematoxylin and eosin and Masson’s trichrome stains. Stained sections were analyzed under light microscope LEICA DMR and imaged using a LEICA DC 300 camera.
Histomorphometric measurements were performed in the NIS-Elements software version 3.2 (Nikon, Tokyo, Japan) on hematoxylin and eosin stained tissue sections. The images were obtained on a microscope Leica DMLS equipped with the camera CMEX-10 Pro (Euromex Microscopen BV, Netherlands) at 100 × magnification. The total area of implants and the total area of blood vessels were measured using the “Annotations and Measurements” tool in the software. The percentage of vascularization (%) was calculated as follows: (total vessel area/total area of implants) × 100.
Histomorphometric measurements were performed in the NIS-Elements software version 3.2 (Nikon, Tokyo, Japan) on hematoxylin and eosin stained tissue sections. The images were obtained on a microscope Leica DMLS equipped with the camera CMEX-10 Pro (Euromex Microscopen BV, Netherlands) at 100 × magnification. The total area of implants and the total area of blood vessels were measured using the “Annotations and Measurements” tool in the software. The percentage of vascularization (%) was calculated as follows: (total vessel area/total area of implants) × 100.
10
Thoracic Aorta Histology and Immunostaining
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Thoracic aortae obtained from DSS rats (NS, HS, and HS+ASA) were fixed in 10% formalin at a pH of 7.4. Dehydration, clarification, and inclusion were performed soon afterward. After blocks were obtained, sections were obtained using a microtome (Leica RM2235; Leica Microsystems) with a thickness of 5 μm. Thoracic aortae were stained with hematoxylin and eosin. The antibodies eNOS (ab76198), vascular cell adhesion molecule‐1 (ab134047), and collagen I (ab96723) were also used for the primary incubation, while fluorescent antibody rabbit antimouse immunoglobulin G and rabbit antimouse immunoglobulin G (A11059 and A11061; both Thermo Fisher Scientific) were included for the second incubation. From each aorta's description, all sections were obtained using a section‐scanning system (Leica SCN400; Leica Microsystems).
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