The femur was decalcified in 10% EDTA (replaced with fresh solution every 3 days) for 3 weeks. Then, the samples were embedded in paraffin and sectioned at 5 μm thick through using a semiautomated rotary microtome (Leica Biosystems RM2245, Nussloch, Germany). The sections were stained by H&E and TRAP kit to examine the number of osteoblast (N.Ob/BS) and osteoclast (N.Oc/BS). All mice were intraperitoneally injected with calcein at 10 and 3 days before euthanasia. The femurs were fixed in 4% paraformaldehyde for 2 days, embedded in methylmethacrylate and sectioned at 50 μm. Fluorescence microscope and ImageJ software were used to analyze the histomorphometric parameter.
Rm2245
Manufactured by Leica
Sourced in Germany, United States, Italy, Japan, France, United Kingdom
The RM2245 is a rotary microtome designed for sectioning paraffin-embedded tissue samples. It features a vertical specimen advance and a precision feed mechanism for accurate and consistent section thickness. The instrument is suitable for a variety of applications in the field of histology and pathology.
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Lab products found in correlation
Tp1020, by Leica (10 mentions)
Technovit 7100, by Kulzer (8 mentions)
Asp300, by Leica (6 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (6 mentions)
Dm4000b, by Leica (5 mentions)
Paraplast plus, by Merck Group (4 mentions)
Paraffin, by Merck Group (4 mentions)
Casecenter 2.9 viewer, by 3DHISTECH (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Omx1200c, by Nikon (3 mentions)
Historesin, by Leica (3 mentions)
Pannoramic scan 150, by 3DHISTECH (3 mentions)
Azure 2, by Merck Group (3 mentions)
Methylene blue, by Merck Group (3 mentions)
Progress c12 plus, by Jenoptik (3 mentions)
Eclipse ci, by Nikon (3 mentions)
Eclipse 50i, by Nikon (3 mentions)
Eclipse ci l, by Nikon (3 mentions)
Dm2500, by Leica (3 mentions)
St5020 cv5030 coverslipper, by Leica (2 mentions)
299 protocols using rm2245
1
Histomorphometric Analysis of Mouse Femurs
2
Tissue Microarray Construction Protocol
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Fixed tissues were sectioned at 5 μm in a standard microtome (Leica Biosystems, RM2245, Barcelona, Spain). Sections were dewaxed in two 10-minutes changes of histoclear and hydrated from 100% alcohol to distilled water through 5-minutes changes of graded alcohols. Then sections were stained 5 minutes in Harris hematoxylin, differentiated in 0.5% hydrochloric acid for 3 seconds and washed with running tap water for 5 minutes. Sections were stained with aqueous eosin solution for 15 minutes and dehydrated through graded alcohols. Finally, samples were cleared with histoclear for 10 minutes (two changes of 5 minutes each) and permanently mounted with DPX mounting medium. Representative areas of the different tissues were selected under a light microscope for tissue array construction using a permanent marker. Afterwards, the hematoxylin-eosin (H&E) stained sections were placed over the donor block for spot selection with the punch of the tissue arrayer. Using the manual Beecher tissue arrayer spots of the array were selected with the 0.6 mm punch and placed in the recipient paraffin block. Once all the spots were placed in the recipient, the block was warmed at 60°C during 10 minutes to melt the new spots with the paraffin of the recipient. 5 μm serial samples of the tissue array were sectioned for further analysis.
3
Myocardial Histological Analysis
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The rats were sacrificed by exsanguination (blood was taken from rats via the right carotid artery). The thoracic cavity was opened and rat hearts were obtained and washed with cold physiological saline. The non-infarcted left ventricular tissue was separated for determination. The non-infarcted left ventricular tissues were fixed in 4% paraformaldehyde and then dehydrated in ascending grades of alcohol and embedded in paraffin, and sliced into 5-µm-thick sections using a microtome (RM2245; Leica Biosystems, Buffalo Grove, IL, USA). The myocardial sections (5-µm-thick) were stained with hematoxylin and eosin (H&E) to analyze the morphological changes in the myocardium.
4
Histological Analysis of Pancreatic and Kidney Tissues
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After fixation, pancreatic and kidney tissues were paraffin-embedded and sectioned at 5µm thickness using a Leica semi-automated RM2245 microtome (Leica Biosystems, Sydney, NSW, Australia). Sections were used for hematoxylin and eosin, Periodic acid-Schiff (PAS), and Masson’s trichrome staining for morphometric analysis. In PAS staining, saccharides will be stained red and the nucleus will be stained blue. Collagen will be stained blue, the nucleus will be stained dark purple, while fiber will be stained red in Masson’s trichrome.
5
Decalcification and Histological Processing
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The right-side samples were immersed immediately in 10% buffer formalin and the decalcified samples were prepared and sectioned by New Histo. Science Laboratory (Tokyo, Japan). Briefly, the specimens were decalcified with 10% EDTA/10 mM Tris-HCl (pH 7.2) for about 2 weeks, dehydrated in a graded series of ethanol and xylene, and then embedded in paraffin. The paraffin-embedded samples were sectioned to 5-μm thickness using a cryostat (RM2245, Leica Biosystems, Wetzlar, Germany). The upper second molar, including the jawbone, was sliced longitudinally along the tooth axis and approximately midway between the buccal and palatal surfaces. All sections were mounted on glass slides and stained with Carazzi’s hematoxylin and eosin after rinsing with running water. Finally, a cover slip was applied to the slide.
6
Histological Analysis of Goat Lung Tissue
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After successful modelling for 24 h, all the goats were fixed on an operating table and sacrificed by bloodletting through the cervical artery. After open the thoracic cavity, the lung was obtained. To ensure the consistency and validation of the samples, lung tissue blocks of the same size and same location in the middle lobe of the right lung were taken and stored in liquid nitrogen for RNA-seq analysis. And another piece of lung tissue in the middle lobe was fixed in 10% (v/v) formaldehyde for one week at room temperature and dehydrated using the Fully Enclosed Tissue Processor (ASP300S, Leica Biosystems, Germany) before embedding in paraffin (EG1150H + C, Leica Biosystems, Germany). The fixed paraffin lung tissues were cut into 5 μm-thick sections using the microtome (RM2245, Leica Biosystems, Germany) and then stained with H&E Staining System (ST5020, Leica Biosystems, Germany) according to the instructions. The sections were visualized with a light microscope (DM4000B, Leica Microsystems, Germany).
7
Penile Smooth Muscle and Collagen Analysis
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The fixed proximal portion of the penis of 6 of 13 sham_SHR and 8 of 12 ESWT_SHR was paraffin embedded and 5-μm sections were performed using a microtome (RM 2245; Leica Biosystems, Nanterre, France). The sections were positioned on SuperFrost plus glass slides (Thermo Fisher Scientific, Waltham, MA, USA), dried, and stained for evaluation of smooth muscle/collagen ratio with Masson’s trichrome method using an automated multistainer (ST5020 &CV5030 Coverslipper; Leica Biosystems). The acquisition of slides images was then performed using Aperio AT2 - Scanner for On-Screen Diagnosis (Leica Biosystems). The percent of smooth muscle and the percent of collagen were computed in the total area of CC from each section by a blinded experimenter using the positive pixel count algorithm (Leica Biosystems). For each animal, 4 transverse sections of penis were stained and analyzed and a mean smooth muscle/collagen ratio was determined.
8
Histomorphometric Analysis of Decalcified Samples
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Samples were fixed in buffered formalin 10% and then decalcified in Osteosoft (Merck KGaA, Darmstadt, Germany). Subsequently, the sample were washed, and the samples were placed in distilled water and then included in paraffin. Sections of about 5 µm thickness were obtained with a microtome (RM2245, Leica Biosystems, Wetzlar, Germany). A central section was stained with scarlet-acid fuchsine and toluidine blue, and then analyzed histomorphometrically.
9
Histological Analysis of Murine Mammary and Gut
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The collected mammary gland and gut tissues of mice were fixed in 4% formaldehyde solution. The tissue was then embedded in paraffin and cut into 5-μm thick sections using a microtome (RM2245, Leica Biosystems, Wetzlar, Germany). The sections were stained with H&E, and histological analysis was performed using an optical microscope (Olympus, Tokyo, Japan). Finally, the pathological score of mammary gland tissue was determined based on previous studies [16 ].
10
Extracting miRNA from FFPE Tumor Samples
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Using a RM2245 semi-automated rotator microtome (Leica Biosystems Nussloch GmbH, Nussloch, Germany), one to three 10 μm sections (depending on tumor size) were cut from FFPE blocks of tumor samples after microdissection to remove surrounding tissue. These sections were transferred to 1.5 mL tubes, and subsequently, miRNA isolation was performed using the miRNeasy FFPE Kit. For subsequent RT-PCR analysis, microRNAs were transcribed utilizing the miScript II RT™ kit. All FFPE miR reagents and qPCR kits were obtained from Qiagen (Hilden, Germany) and used according to the manufacturer’s instructions.
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