After FAA fixation, the samples were dehydrated according to the methods of a previous study [17 (link)–19 (link)], processed with three changes of 100% paraffin at 63 °C, and finally embedded. The paraffin-embedded material was cut into 10-mm-thick sections (RM2255 Fully Automated Rotary Microtome; Leica, Germany), and the sections were stained with Safranin O and fast green FCF (Sigma-Aldrich, USA) [17 (link)]. The slices were observed and photographed using a Nikon D3000 camera, a Leica M205 FA fluorescence stereo microscope, and a Leica DM 6000B fully automated upright microscope (Leica Microsystems GmbH, Wetzlar, Germany).
Rm2255 fully automated rotary microtome
Manufactured by Leica
Sourced in Germany, United States
The RM2255 Fully Automated Rotary Microtome is a laboratory equipment designed for the sectioning of biological samples. It features automated operation and precise control of section thickness.
Automatically generated - may contain errors
Lab products found in correlation
Fast green fcf, by Merck Group (2 mentions)
Dm6000b, by Leica (2 mentions)
Safranin o fast green counterstaining, by Merck Group (2 mentions)
Safranin o, by Merck Group (1 mentions)
Safranine o, by Merck Group (1 mentions)
Goat poly, by Santa Cruz Biotechnology (1 mentions)
Tp1020 tissue processor, by Leica (1 mentions)
Picrosirius red, by Zeiss (1 mentions)
Picrosirius red, by Abcam (1 mentions)
Masson s trichome, by Abcam (1 mentions)
Axio observer, by Zeiss (1 mentions)
Dm750 microscope, by Leica (1 mentions)
Eosin solution, by Merck Group (1 mentions)
Mayer s haematoxylin solution, by Merck Group (1 mentions)
Imager m2 microscope, by Zeiss (1 mentions)
Zen 2, by Zeiss (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
Tp1020, by Leica (1 mentions)
Eg1150, by Leica (1 mentions)
Axiostar plus 1169 149, by Zeiss (1 mentions)
11 protocols using rm2255 fully automated rotary microtome
1
Paraffin Embedding and Tissue Staining
2
Histological Analysis of Samples
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For histological analysis, the samples were immersed in FAA fixative and placed under vacuum at 4 °C. Samples were dehydrated in gradient ethanol and then embedded in paraffin. Ten-mm-thick sections (RM2255 Fully Automated Rotary Microtome; Leica, Germany) were stained with Safranine O and fast green FCF (Sigma-Aldrich, USA). The slices were observed and photographed using a Leica DM 6000B fully automated upright microscope (Leica Microsystems GmbH, Wetzlar, Germany).
3
Immunohistochemical Analysis of Tumor Markers
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The efficacy of BSM-0004 and oncogenic expression of tumour marker Ki67 and hCA-IX were evaluated by immunohistochemistry of tumour samples as described in the previous study51–54 (link). Briefly, tumour tissues were fixed with 4% paraformaldehyde then embedded with high grade of paraffin. The tissue-sections were cut in 5 µm thick using a microtome (Leica RM2255 Fully Automated Rotary Microtome, Wetzlar, Germany), then sliced tissues were fixed onto glass slides and deparaffinised with various percentage of xylene subjected to rehydrated with different concentrations of alcohol (C2H5OH). All slides were incubated with methanol containing 3% of hydrogen peroxide to be retrieved. After blocking with 5% horse serum, the slides were incubated with rabbit polyclonal antibodies for Ki67 (diluted, 1:400; Goat poly, Santa Cruz, Santa Cruz, CA) and anti-CA-IX (1:500 diluted, Mouse, abcam ab107257, Cambridge, UK). The immunohistopathological expression of Ki67 and hCA-IX was counter stained with biotinylated anti-rabbit and anti-mouse IgG (Vector Laboratories, Burlingame, CA). The diaminobenzidine (DAB) substrate was used to develop and precipitate the protein. Lastly, Mayer’s haematoxylin blue staining was performed to distinctly observe nuclear portion. The Ki67 and hCA-IX protein levels were detected using Leica microscope system (Wetzlar, Germany).
4
Histopathological Analysis of Spleen Samples
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Tissue for histopathology was processed as described in [21, 22] . Brie y, spleen samples were processed for dehydration, clearing, and impregnation by Leica TP1020 Tissue Processor. Para n blocks were prepared using ThermoFisher Histostar Tissue Embedding Station and serial sections of 3 µm thickness were cut using Leica RM2255 Fully Automated Rotary Microtome. Sections were placed on slides with 50 mM ethanol, then immerged in dissolved 0.1% gelatin. Dewaxing was performed by emerging the prepared slides 2 × 5 min in Xylol. Samples were then washed 3 × 2 min with 95% ethanol, then for 2 min with 75% ethanol, then 50% ethanol. Samples were drained for 3 min with water, treated with 0.37% HCL in 70% ethanol to remove hemotoxylin excess, and redrained for 2 min with water. Nucleus was stained using ammonia, and cytoplasm was stained using eosin. Slides were washed 5 × 10 min with 95% ethanol, emerged in Xylol and mounted with coverslips. Mounted tissue sections were observed under Swift M2250 Series Monocular lab light microscope for structural changes and abnormalities.
5
Evaluating Right Ventricular Hypertrophy
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After measuring RVSP, the pulmonary circulation was flushed with phosphate buffered saline (PBS) from the right ventricle, then the heart and lungs were removed. The right ventricle was carefully separated from the heart and weighed. Right ventricular hypertrophy was assessed by calculating the ratio of the weight of right ventricle (RV) to the weight of the left ventricle (LV) and septum (S) [RV/(LV + S)].
The left lungs were fixed for 24 h in 10% neutral formalin solution, and the right lungs were placed in liquid nitrogen for protein and RNA extraction. After paraffin embedding, the slides (6 μm thickness) were sectioned using Leica RM2255 Fully Automated Rotary Microtome (Leica) and were stained for morphological analysis.
H&E and immunohistochemical staining was performed according to the manufacturer's instructions. Additional details are described in Supporting Information.
The left lungs were fixed for 24 h in 10% neutral formalin solution, and the right lungs were placed in liquid nitrogen for protein and RNA extraction. After paraffin embedding, the slides (6 μm thickness) were sectioned using Leica RM2255 Fully Automated Rotary Microtome (Leica) and were stained for morphological analysis.
H&E and immunohistochemical staining was performed according to the manufacturer's instructions. Additional details are described in Supporting Information.
6
Histopathological Analysis of Liver Samples
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The liver samples were fixed in 4% paraformaldehyde at room temperature and then embedded in paraffin. Tissue fragments were cut into 4 μm thick sections using a Leica RM2255 Fully Automated Rotary Microtome (Leica, Wetzlar, Germany) and stained with hematoxylin and eosin (H&E). Hepatic inflammation was evaluated by recording inflammatory cell infiltration and steatosis.
7
Histological Analysis of Sponge Body Structure
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Specimens were fixed overnight by 4% formaldehyde in seawater at 4 °C. Histological sections specimens comprising the sponge’s whole body, thus including the ectosome (outer cortex) and choanosome (inner part), were embedded in paraffin wax and cut on a Leica RM2255 Fully Automated Rotary Microtome (Leica, Wetzlar, Germany) at 10 μm. Cross-transversal paraffin sections were stained with Hematoxylin-eosin (H&E) for tissues’ general overview and with Masson’s trichome and Picrosirius Red (Abcam, Cambridge, UK) for specific collagen observation [62 (link),63 (link)]. Masson’s trichome stains collagen blue and cytoplasm red; Picrosirius Red stains collagen green, red, or yellow under polarized light, depending on fiber thickness and packing [62 (link),64 (link)]. H&E and Masson’s trichome-stained semi-thin sections were observed under a Leica DM750 microscope (Leica, Wetzlar, Germany), and Picrosirius Red stained semi-thin sections were observed with an Axio Observer (Carl Zeiss, Jena, Germany) under polarized light.
8
BAT Tissue Preparation and Histological Analysis
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BAT was dissected from 21-week-old male mice on a HFD. It was post-fixed in 4% PFA at 4 °C for more than 16 h and embedded with pre-set program in ShandonTM ExcelsiorTM ES Tissue Processor Accessories (ThermoFisher Scientific). In general, tissue was put in formalin for 1 h twice, 70% ethanol for 2 h, 96% ethanol for 2 h twice, 100% ethanol for 1 or 2 h three times, xylene for 1 or 2 h three times, and liquid wax for 1 h to 1.67 h three times, at 45 °C. Tissues were then embedded in blocks using Leica EG1150 H Heated Paraffin Embedding Module and Leica EG1150 C Cold Plate for Modular Tissue Embedding System. Sample blocks were stored at RT until cutting. Slices (5 µm) were cut with Leica RM2255 Fully Automated Rotary Microtome. Slices were deparaffinized with xylene for 20 min, put into isopropanol for 2 min, gradually hydrated with diluted ethanol and water, stained with Mayer´s haematoxylin solution (Sigma-Aldrich) followed by eosin solution (Sigma-Aldrich) after washing, washed in water, gradually dehydrated in diluted ethanol, put in xylene and finally mounted with Cytoseal XYL (ThermoFisher Scientific). Slides were stored at RT, and imaged with Zeiss Imager M2 microscope and the software Zen 2 (Carl Zeiss AG).
9
Histopathological Assessment of Organ Tissues
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Organs (liver, kidney, lung, spleen, heart, brain, ovary and testis) from different groups were fixed in neutral buffered 10% v/v formalin solution for histopathological assessment. After fixation, the organs were dehydrated in graded alcohol (TP1020, semi-enclosed benchtop tissue processor, Leica Biosystems, Germany), embedded in paraffin (EG1130 and EG1150, cold plate for cooling embedding molds and paraffin blocks and modular tissue embedding center, Leica Biosystems, Germany), sectioned into 4 µm thick (RM2255, fully automated rotary microtome, Leica Biosystems, Germany) and stained with Weigert’s Hematoxylin and Eosin (Suvarna et al., 2012 ). Visualization and microphotographs were captured under a light microscope (Axiostar plus 1169-149, Carl Zeiss, Germany) at magnification 10×.
10
Safranin O/Fast Green Staining for GAGs
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Safranin O/Fast green staining was used to assess glycosaminoglycans (GAGs) content. Samples were fixed in 10% buffered formalin phosphate solution (Fisher Chemical, Fair Lawn, NJ, United States) overnight and dehydrated with an increasing ethanol gradient. After being soaked in xylene for 2 h and then in liquid paraffin overnight, samples were embedded in paraffin. The blocks were sectioned at 6 μm thickness using the RM 2255 Fully Automated Rotary Microtome (Leica Biosystems, Buffalo Grove, IL, United States). Slides were dried and subjected to Safranin O/Fast green counterstaining (Sigma-Aldrich, St. Louis, MO, United States). Cell nuclei were stained with Hematoxylin solution (Sigma-Aldrich).
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