For histological examinations, a vertical incision was made just below the right lobe of the liver. The liver sample was washed and placed in 10% formalin for a week. For tissue preparation, the sample was dehydrated in ascending concentrations of ethanol, cleared by xylene solution, and then embedded in paraffin. Thin 4-μm sections were sliced using a microtome (Leica RM 2125, Germany) and stained with H and E staining. Images were captured by the 40x objective lens. In the images, the full cellular area, hepatocyte outline, and maximum and minimum axes of each hepatocyte (to obtain the mean axis) were measured. At least 50 hepatocytes from each zone were assessed in each liver. A separate measurement diameter of central hepatic vein (CHV) was performed using the same assay too. The histological data were collected using an Olympus BX-51T-32E01 research microscope (Leica RM 2125, Nussloch, Germany) connected to a DP12 Camera (Olympus Optical, Tokyo, Japan) with a 3.34-million pixel resolution and Olysia Bio-software (Olympus Optical Co. LTD, Tokyo, Japan).[ 11 ]
Rm2125
Manufactured by Leica
Sourced in Germany, United States, United Kingdom, Italy
The RM2125 is a rotary microtome designed for sectioning paraffin-embedded tissue samples. It features a motorized drive system and a user-friendly control panel for precise adjustment of section thickness. The device is intended for use in histology and pathology laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Olysia bio software, by Olympus (9 mentions)
Bx 51t 32e01 research microscope, by Olympus (5 mentions)
Antigen retrieval solution, by Abcam (4 mentions)
Dp12 camera, by Olympus (3 mentions)
Paraplast x tra, by Thermo Fisher Scientific (3 mentions)
Tunel kit, by Roche (3 mentions)
Axiocam erc5, by Zeiss (3 mentions)
Eclipse 200, by Nikon (3 mentions)
Dm5000, by Leica (3 mentions)
Tp1020, by Leica (3 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Morgagni 268d, by Thermo Fisher Scientific (2 mentions)
Light microscopy, by Olympus (2 mentions)
Light microscopy, by Leica (2 mentions)
Primo star, by Zeiss (2 mentions)
Bx 51t 32e01, by Olympus (2 mentions)
Mayer s hematoxylin, by Merck Group (2 mentions)
Eg1160, by Leica (2 mentions)
Ant software, by Bruker (2 mentions)
Evos fl auto 2 imaging system, by Thermo Fisher Scientific (2 mentions)
131 protocols using rm2125
1
Histological Assessment of Hepatocytes
2
Cartilage Histology: Formalin Fixation, Decalcification, and Safranin O Staining
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The human cartilage explants were fixed in 10% formalin (MilliporeSigma) for 72 hours. The specimens were decalcified in Richman-Gelfand-Hill solution, processed in a Tissue-Tek VIP 1000 tissue processor (Miles Laboratories, USA), and embedded in a single block of Paraplast X-tra (Thermo Fisher Scientific, USA). Blocks were trimmed to expose the tissue using a rotary microtome (Leica RM2125; Leica Microsystems, Germany). Ten adjacent sections were collected at intervals of 0 μm, 100 μm, and 200 μm. Two serial 6 μm-thick sections from each interval were stained with Safranin O.
In the in vivo study, the femora and tibiae of SD rats were hemisected in the midsagittal plane, and each half was embedded in a single block of Paraplast X-tra. Blocks were trimmed to expose the cartilage. Ten adjacent sections were collected at intervals of 0 μm, 100 μm, and 200 μm (Leica RM2125). Two serial 6 μm-thick sections from each interval were stained with Safranin O.
In the in vivo study, the femora and tibiae of SD rats were hemisected in the midsagittal plane, and each half was embedded in a single block of Paraplast X-tra. Blocks were trimmed to expose the cartilage. Ten adjacent sections were collected at intervals of 0 μm, 100 μm, and 200 μm (Leica RM2125). Two serial 6 μm-thick sections from each interval were stained with Safranin O.
3
Cartilage Explant Analysis with Ipriflavone
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Seventy-two hours after culture with ipriflavone or 0.1% DMSO, the human cartilage explants were fixed in 10% formalin (Sigma Aldrich, St Louis, MO, USA) for 72 h. The specimens were decalcified in Richman-Gelfand-Hill solution, processed in a Tissue-Tek VIP 1000 tissue processor (Miles, Elkhart, IN, USA), and embedded in a single block of Paraplast X-tra (Thermo-Fisher, Hampton, NH, USA). Blocks were trimmed to expose the tissue using a rotary microtome (Leica RM2125, Leica Microsystems Ltd., Wetzlar, Germany). Ten adjacent sections were collected at intervals of 0 μm, 100 μm, and 200 μm. Two serial 6-μm-thick sections from each interval were stained with Safranin O.
In the in vivo study, the femurs and tibiae were hemisected in the midsagittal plane, and each half was embedded in a single block of Paraplast X-tra (Thermo-Fisher, Hampton, NH, USA). Blocks were trimmed to expose the cartilage. Ten adjacent sections were collected at intervals of 0 μm, 100 μm, and 200 μm (Leica RM2125, Leica Microsystems Ltd., Wetzlar, Germany). Two serial 6-μm-thick sections from each interval were stained with Safranin O. Cartilage degradation was quantified by two independent and blinded observers using an Osteoarthritis Research Society International (OARSI) grading system [27 (link)].
In the in vivo study, the femurs and tibiae were hemisected in the midsagittal plane, and each half was embedded in a single block of Paraplast X-tra (Thermo-Fisher, Hampton, NH, USA). Blocks were trimmed to expose the cartilage. Ten adjacent sections were collected at intervals of 0 μm, 100 μm, and 200 μm (Leica RM2125, Leica Microsystems Ltd., Wetzlar, Germany). Two serial 6-μm-thick sections from each interval were stained with Safranin O. Cartilage degradation was quantified by two independent and blinded observers using an Osteoarthritis Research Society International (OARSI) grading system [27 (link)].
4
Histological Analysis of Rat Hearts
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Immediately after the sacrifice of the rats, the hearts were removed, washed with iced normal saline, fixed in 10% formalin, and decalcified with formic acid (31.5% formic acid and 13% sodium citrate). The hearts were embedded in paraffin for sectioning by standard histological methods [13 (link)]. Sections (4 μm, Leica RM2125, Germany) from the left ventricle were stained with hematoxylin and eosin (H&E) and examined by light microscopy (Leica DMR, Germany) at 200x magnification.
5
Paraffin Embedding and Trichrome Staining
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All embryos for paraffin histology were dehydrated into 100% ethanol (or changed from 100% methanol to 100% ethanol) before clearing in Histosol (National Diagnostics). Embryos were washed three times for 20 min each in Histosol at room temperature, twice for 30 min each in 1:1 Histosol:paraffin at 60°C and then then moved into molten paraffin wax for overnight at 60°C. The following day, embryos were washed four times for 1 h each in paraffin, and then embedded in peel-a-way moulds. Embedded embryos were sectioned at 8 μm on a Leica RM2125 rotary microtome and sections were mounted on Superfrost Plus slides (Fisher). Histochemical staining was performed using the modified Masson's trichrome staining protocol of Witten and Hall (2003) (link).
6
Histopathological Analysis of Ankle Synovial Tissue
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The abovementioned left ankle joints (see 2.3.3) were immediately fixed in the 10% formalin, and decalcified with 15% ethylenediaminetetraacetic acid (EDTA) for 30 days. After which, the same tissue was dehydrated with different concentrations of ethanol step-by-step, embedded in paraffin, sliced (using a Leica RM2125, Buffalo Grove, United States), and stained with hematoxylin and eosin (HE). Pathological features of the synovial tissue were observed under an optical microscope, especially tissue involving inflammatory cell infiltration and hyperplasia of the synovial tissue, pannus, and cartilage erosion.
7
Histological Analysis of Submandibular Glands
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The histological analysis in submandibular glands were performed only in animals described in protocol 1 (Fig 1 ). Submandibular glands were fixed in 10% buffered formalin. Subsequently, these glands were dehydrated in alcohol (80, 90 and 100%), cleared in xylene and embedded in paraffin. Histological sections with a thickness of 5 μm were acquired using a microtome (Leica RM2125). Then these sections were placed on slides and stained with hematoxylin and eosin. Histological slides were examined and micrograph pictures were obtained using an optical microscope (Olympus BX41).
8
Histological Analysis of Murine Skin
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The dorsal skin was harvested from three random mice from each group and stored in 10% (v/v) formaldehyde. Skin samples were dehydrated through a graded series of ethanol baths, embedded in paraffin, and sectioned (4 μm thick) using a microtome (RM2125, Leica, Germany). The sections were then stained with hematoxylin and eosin (H&E) and Picrosirius red (Junqueira et al. [1979]) [15 (link), 16 (link)]. Epithelial thicknesses were measured, and areas of collagen were imaged using a microscope (BX53, Olympus, Japan) running cellSens software (Olympus). Collagenous areas were measured with the aid of ImageJ software (National Institutes of Health, USA) [17 ].
9
Histological Analysis of Nerve Structure
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The samples were embedded in paraffin after fixation in 10% buffered formalin for 24 hours. Then, 4 µm thick longitudinal sections along the neural structures or transverse sections from proximal, middle and distal areas of the specimens were obtained using a microtome (Leica RM2125, Wetzlar, Germany). After deparaffinisation and serial dehydration with ethanol, the sections were stained with toluidine blue and Masson's trichrome. Two blinded investigators using cross sections performed the quantitative histological measurements. Five random fields at X100 magnification were chosen from proximal, middle, and distal segments. The images were captured by a digital camera connected to a light-microscope (Leica DM3000, Wetzlar, Germany) and computer. Two blinded investigators made all measurements. The number of axons per 1 mm2 was estimated and axon diameter and myelin sheath thickness were measured on each sample using an image software program (Neurolucida software, MBF Bioscience, Williston, VT, USA).
10
Histopathological and Ultrastructural Evaluation of Heart Tissue
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For histopathological evaluation, heart tissues fixed in 10% neutral buffered formalin were embedded in paraffin to make tissue blocks. The blocks were then cut into 5 μm thick histologic section with microtome (Leica RM 2125, Germany). The sections were then stained with hematoxylin and eosin (H&E). At least three hearts from each group were examined under light microscope (Dewinter technologies, Italy) for any pathological changes. The findings were reported as (−) no change; (+) focal change; (++) patchy change; (+++) confluent change.
For ultrastructural evaluation heart tissues stored in Karnovsky’s fixative were washed with ice-chilled phosphate buffer (0.1 M, pH 7.4) and then fixed with 1% osmium tetroxide at 4 °C. Following this, tissues were embedded in araldite CY212 and tissue blocks were made. Ultrathin sections of 70–80 nm thickness were cut using an ultramicrotome (Ultracut E, Reichert, Austria) and stained with uranyl acetate and lead acetate. The sections were then visualized under transmission electron microscope (Morgagni268D, FeiCo., The Netherlands).
For ultrastructural evaluation heart tissues stored in Karnovsky’s fixative were washed with ice-chilled phosphate buffer (0.1 M, pH 7.4) and then fixed with 1% osmium tetroxide at 4 °C. Following this, tissues were embedded in araldite CY212 and tissue blocks were made. Ultrathin sections of 70–80 nm thickness were cut using an ultramicrotome (Ultracut E, Reichert, Austria) and stained with uranyl acetate and lead acetate. The sections were then visualized under transmission electron microscope (Morgagni268D, FeiCo., The Netherlands).
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