Mouse sciatic nerves were dissected and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Nerve samples were then osmicated, dehydrated, and embedded in araldite resin. Transverse nerve sections (1 µm) were cut on a Leica RM2065 microtome and stained with methylene blue Azure II. Images were collected on a Leica DMR microscope or an Olympus BX61 microscope. Axon numbers were determined from two non-overlapping fields (50×50 µm) from each of three mutant and three control nerve samples. Axon diameters were measured by Image J.
Rm2065 microtome
Manufactured by Leica
Sourced in Germany
The RM2065 is a manual rotary microtome designed for precision sectioning of paraffin-embedded tissue samples. It features a robust construction, a coarse and fine feed mechanism, and a range of specimen clamps to accommodate various sample sizes. The RM2065 enables the user to obtain high-quality sections for a variety of histological applications.
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Lab products found in correlation
Dmr microscope, by Olympus (2 mentions)
Bx61 microscope, by Olympus (2 mentions)
Osmic acid, by Ted Pella (2 mentions)
Araldite resin, by Ted Pella (2 mentions)
Epon 812, by Ted Pella (1 mentions)
Xl30 feg, by Philips (1 mentions)
Bh 2 light microscope, by Olympus (1 mentions)
Silverstar taq dna polymerase, by Eurogentec (1 mentions)
Bouin s solution, by Merck Group (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Fresenius (1 mentions)
Excelsior tissue processor, by Thermo Fisher Scientific (1 mentions)
11 protocols using rm2065 microtome
1
Quantitative Histological Analysis of Mouse Sciatic Nerve
2
Glutaraldehyde Fixation and Electron Microscopy
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Samples were washed three times with PBS, fixed in 2.5% glutaraldehyde for 60 min, and stained with 1% osmic acid for 1 h. After dehydrating through a graded ethanol series, samples were embedded in Epon overnight at 60 °C and polymerized for 48 h. Ultrathin sections were cut on an RM2065 microtome (Leica) and examined under an electron microscope (CM 10; Philips, Eindhoven, Holland).
3
Ultrastructural Evaluation of Remyelination
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All three scaffolds (DON, DSN and CS) in vitro were coated with gold and examined by scanning electron microscopy (Philips XL30 FEG). For ultrastructural observation of remyelination under a transmission electron microscope (Philips CM 10), the in vitro DRG cultures and the in vivo spinal cord segments containing a functional scaffold were post-fixed in fixative solution containing 2.5% glutaraldehyde and 4% paraformaldehyde overnight at 4°C. The samples were then washed in PBS, dehydrated through a graded ethanol series, and flat-embedded in Epon812 (TED PELLA, Redding, CA, USA). For in vivo quantification, semi-thin sections of spinal cord segments were cut using a Leica RM2065 microtome, stained with toluidine blue (5% in a borax solution), and mounted on glass slides. Three semi-thin sections were selected from the injury/graft site of the spinal cord of each rat for remyelination analysis. Nine sections were selected from each group of three animals. Three regions of interest were selected in each section and examined at 1000× magnification. Newborn myelin sheaths (thin sheaths and light in color) were counted and compared between the two groups. Ultrathin sections (100 nm in thickness) of the DRG cultures and the injury/graft site of the spinal cord were cut, double-stained with lead citrate and uranyl acetate, and examined under the transmission electron microscope.
4
Lignin Detection in Flax Tissues
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Flax stems, roots and leaves were fixed with 4% paraformaldehyde in phosphate buffer (0.2 M, pH 7) for 16 h. They were then washed in phosphate buffer containing 0.1 M glycine and dehydrated through a series of ethanol solutions and progressively embedded in paraffin (ParaplastPlus; Oxford Labware). Tissue sections were obtained with a Leica RM2065 microtome and placed on silanised-coated slides. Before staining, tissue sections were deparaffinised with Histoclear (Labonord) and rehydrated with decreasing concentrations of ethanol. The presence of lignin was determined by staining with the Wiesner reagent (phloroglucinol–HCl) [83 (link)].
5
Stem Morphometry and Raman Analysis
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Stem samples (1-cm-long) were collected from the middle region of the stem (10 cm above the cotyledon scar) from ten individual control and ten individual stressed plants. For morphometric analyses samples were fixed in EtOH (70% w/v) for a minimum of three days at 4°C before being dehydrated through a graded EtOH/Histoclear series and embedded in Paraplast Plus. Stem transversal sections (10 μm) were obtained with a Leica RM65 microtome equipped with an S65 metal blade. Sections were mounted on glass slides and stained with Toluidine Blue-O (TBO) prior to observation with an Olympus BH-2 light microscope. For Raman microspectroscopy, samples were embedded in PEG (polyethylene glycol) 1,500, as previously described (Gierlinger, 2014 (link)). Transversal sections (30 μm) were obtained with a Leica RM2065 microtome for Raman focusing. PEG was removed by multiple water baths and sections were air dried prior to mounting in bidistilled water with a coverslip sealed by nail polish to avoid desiccation. Standard glass slides (superfrost™) and glass coverslips were used.
6
Histological Analysis of Tracheal Epithelium
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After the last measurement of MCT and CBF, an epithelial sample of the trachea was collected and fixed with 4% formalin-buffered solution for routine histological processing. Three animals were analyzed from each group for qualitative analysis of the tissue sample obtained at T180. These fragments were dehydrated in an ethanol gradient (70° to 100°), cleared with xylol and blocked in paraffin. Five-micrometer sections were obtained using a Leica RM2065 microtome. The sections were deparaffinized with xylol, hydrated in an ethanol gradient (100° to 70°) and dyed for 2 minutes with Harris’s hematoxylin. The sections were then washed in running water and counterdyed with eosin for 15 minutes. They were then washed in running water, dehydrated in an ethanol gradient (95° to 100°), cleared with xylol and mounted with laminule and entellan to analyze the histopathological alterations. The samples were qualitatively analyzed with an optical microscope by a pathologist blind to the group allocation.
7
Quantitative Histological Analysis of Mouse Sciatic Nerve
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Mouse sciatic nerves were dissected and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Nerve samples were then osmicated, dehydrated, and embedded in araldite resin. Transverse nerve sections (1 µm) were cut on a Leica RM2065 microtome and stained with methylene blue Azure II. Images were collected on a Leica DMR microscope or an Olympus BX61 microscope. Axon numbers were determined from two non-overlapping fields (50×50 µm) from each of three mutant and three control nerve samples. Axon diameters were measured by Image J.
8
Fetal Gonad Sex Determination
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Fetal gonads were fixed in 4% paraformaldehyde (PFA) (MERCK, Darmstadt, Germany) overnight (o/n) at 4°C and adult ovarian pieces were fixed either in Bouin’s solution (Sigma-Aldrich, St. Louis, USA) or 4% PFA o/n at 4°C. Thereafter, the tissues were washed 3× in phosphate-buffered saline (PBS), transferred to 70% ethanol solution and embedded in paraffin using a Shandon Excelsior tissue processor (Thermo Scientific, Altrincham, UK). The material was sectioned (5 μm) using a RM2065 microtome (Leica Instruments GmbH, Wetzlar, Germany) and mounted using Prolong Gold (Life Technologies, Carlsbad, USA) on StarFrost slides (Waldemar Knittel, Braunschweig, Germany). The sex of the human fetal gonads was determined using a genomic PCR for AMELOGENIN (FW 5′ CTG ATG GTT GGC CTC AAG CCT GTG 3′ and RV 5′-TAA AGA GAT TCA TTA ACT TGA CTG-3′), that resulted in two different sized amplicons when from the X (977 bp) or Y (790 bp) chromosomes [28 (link)]. The PCR was performed using SilverStar Taq DNA polymerase (Eurogentec, Seraing, Belgium) with a PCR cycle of 95°C for 5 minutes, 34 times 95°C for 1 minutes, 60°C for 30 seconds, 72°C for 2 minutes and a final extension step at 72°C for 10 minutes. The PCR products were run on a 1.5% agarose gel.
9
Sciatic Nerve Ultrastructural Analysis
10
Sciatic Nerve Ultrastructural Analysis
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Sciatic nerve segments were excised and post-fixed for 48 h at 4 °C using 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Specimens were washed using phosphate buffer, post-fixed with 1 % osmic acid (Ted Pella), dehydrated in graded (30–100 %) ethyl alcohol and propylene oxide, and embedded in Araldite resin (Ted Pella). One-micron-thick sections were cut using a diamond knife in an automated RM2065 microtome (Leica Microsystems) and stained using methylene blue/azure II solution [38 (link)]. Sections from three animals per group and three randomly selected areas per section were analyzed.
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