The liver, lungs, kidneys, intestine, and brains were excised from the animals after the indicated exposure times. Tissue samples for hematoxylin and eosin (H&E), Thioflavin T, or immunofluorescence staining (see below) were embedded in polyfreeze tissue freezing medium (OCT; Polysciences, Inc., Warrington, PA, USA) and immediately frozen in liquid nitrogen. Cryosections (7 µm) were obtained with a Leica CM1850 cryotome. At least 10 samples for each tissue were scored for MWCNT aggregates by counting the aggregates found in five blinded fields for each H&E stained slide using a light microscope. Samples for transmission electron microscopy (TEM) were fixed in 4% glutaraldehyde, washed in 0.1 M cacodylate buffer pH 7.4, and postfixed with 1% osmic acid in cacodylate buffer, pH 7.4. After standard dehydration in ethanol series, samples were embedded in an Epon–Araldite 812 mixture and sectioned with a Reichert Ultracut S ultratome (Leica Microsystems, Wetzlar, Germany). Semithin sections were stained by conventional methods (crystal violet and basic fuchsin) and observed with a light microscope (Nikon Instruments, Melville, NY, USA). Thin sections were stained by uranyl acetate and lead citrate and observed with a JEOL 1010 electron microscope (JEOL, Tokyo, Japan).
Cm1850 cryotome
Manufactured by Leica
Sourced in Germany
The Leica CM1850 is a cryotome, a specialized laboratory instrument used for the preparation of thin tissue sections for microscopic examination. It is designed to precisely cut frozen tissue samples into thin slices, enabling detailed analysis and observation of the tissue's structural and cellular components.
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Lab products found in correlation
Tissue tek oct compound, by Sakura Finetek (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Stempro chondrogenesis differentiation kit, by Thermo Fisher Scientific (2 mentions)
1010 electron microscope, by JEOL (1 mentions)
Col3a1, by Abcam (1 mentions)
Phosphorylated stat3, by Cell Signaling Technology (1 mentions)
Mmp 13, by Thermo Fisher Scientific (1 mentions)
Notch1 primary antibody, by Santa Cruz Biotechnology (1 mentions)
Col2a1, by Thermo Fisher Scientific (1 mentions)
Col1a1, by Abcam (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
Rabbit anti human ki67, by Abcam (1 mentions)
D p x neutral mounting medium, by Merck Group (1 mentions)
Diaminobenzidine dab, by Agilent Technologies (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Saturated picric acid, by Electron Microscopy Sciences (1 mentions)
Ethd 1, by Thermo Fisher Scientific (1 mentions)
Axiocam mrc5 camera, by Zeiss (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
21 protocols using cm1850 cryotome
1
Histological Analysis of MWCNT Exposure
2
Meibomian Gland Immunostaining Protocol
3
Histological Analysis of Knee Joints
4
Ultrastructural Analysis of Leech Tissues
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This technique is utilized to highlight the three-dimensional architecture of extracellular and intracellular areas.
SEM analysis: samples of leech body cross-sections, were embedded in Polyfreeze tissue freezing medium (OCT, Polyscience, Eppelheim, Germany) and immediately frozen in liquid nitrogen. Cryosections (8–10 μm) were obtained with a Leica CM1850 cryotome and collected on gelatine-coated slides.
After washing in phosphate-buffered saline (PBS) (pH 7.2) for 5 min at room temperature, were postfixed in a solution of 1% osmium tetroxide and 1.25% potassium ferrocyanide for 30 min at room temperature. After washing in PBS (pH 7.2) for 5 min at room temperature, slides were immersed in 0.1% osmium tetroxide in PBS for 48 h at room temperature then processed for SEM observation.
TEM analysis: samples from cross-sections of leech body were fixed in a solution of 1% osmium tetroxide and 1.25% potassium ferrocyanide for 30 min at room temperature. After washing in PBS (pH 7.2) for 5 min at room temperature, specimens were immersed overnight in 0.1% osmium tetroxide in PBS at room temperature then processed for TEM observation.
SEM analysis: samples of leech body cross-sections, were embedded in Polyfreeze tissue freezing medium (OCT, Polyscience, Eppelheim, Germany) and immediately frozen in liquid nitrogen. Cryosections (8–10 μm) were obtained with a Leica CM1850 cryotome and collected on gelatine-coated slides.
After washing in phosphate-buffered saline (PBS) (pH 7.2) for 5 min at room temperature, were postfixed in a solution of 1% osmium tetroxide and 1.25% potassium ferrocyanide for 30 min at room temperature. After washing in PBS (pH 7.2) for 5 min at room temperature, slides were immersed in 0.1% osmium tetroxide in PBS for 48 h at room temperature then processed for SEM observation.
TEM analysis: samples from cross-sections of leech body were fixed in a solution of 1% osmium tetroxide and 1.25% potassium ferrocyanide for 30 min at room temperature. After washing in PBS (pH 7.2) for 5 min at room temperature, specimens were immersed overnight in 0.1% osmium tetroxide in PBS at room temperature then processed for TEM observation.
5
Histological and Immunohistochemical Analysis
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For histological analysis, cryosections (6–10 µm thick) were prepared using a Leica CM1850 cryotome (Wetzlar, Germany). Sections were collected onto warm, charged Menzel Superfrost slides (Thermo Fisher), fixed in ice-cold 100% acetone, air dried and stored at −80 °C. For immunohistochemistry, tissues were fixed in paraformaldehyde and dehydrated through a graded series of ethanol and xylene, before being embedded in paraffin. Sections (5 µm) were mounted on to glass Menzel Superfrost slides (ThermoFisher Scientific). Immunohistochemistry was performed using antibodies for the proliferation marker Ki67 (rabbit anti-human Ki67, Abcam, Cambridge, UK) and for the infiltration of murine blood vessels using rabbit anti-murine CD31 antibody (Abcam). Tissue sections were incubated with HRP-polymer conjugates (SuperPicture, Thermo Fisher Scientific), and incubated with the chromagen diaminobenzidine (DAB) (Dako, Glostrup, Denmark), as per manufacturer’s specifications. Slides were counterstained with Mayer’s hematoxylin, dehydrated, and mounted with coverslips using D.P.X neutral mounting medium (Sigma Aldrich). All sections were counterstained with Mayer’s hematoxylin (Sigma Aldrich) and mounted with coverslips using D.P.X with Colourfast (Fronine, ThermoFisher Scientific).
6
Bmp3 Protein Localization via LacZ Reporter
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To examine the tissue localization of the Bmp3 protein, the β-galactosidase (LacZ) reporter protein was used. Mice at P0 (bred from WT or Bmp3−/− parents exclusively) were terminated using deep ether anesthesia and tissue samples were fixed in 10% paraformaldehyde. Samples were then cryoprotected for three days in phosphate-buffered saline solutions of increasing sucrose concentrations (10%, 20%, and 30%), after which they were embedded in OCT Tissue Tek (Sakura, Osaka, Japan) and frozen at −80 °C. Frozen samples were then transferred to −20 °C and prepared for cutting on a CM1850 cryotome (Leica, Wetzlar, Germany) at a thickness of 20 µm. Cross-sections were stained using a standard procedure for X-gal [50 (link)] or standard H&E staining. Samples were imaged using a BX-53 microscope (Olympus, Tokyo, Japan).
7
Mouse Brain Tissue Preparation for Proteomics
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Details about the tissue preparation can be found in a previous study [25 (link)]. Mouse brain tissue was dissected, snap frozen (−80 °C) and stored before use. A thick slice was then cut from it and fixed overnight at 4 °C in 4% paraformaldehyde (PFA). It was subsequently embedded using Tissue- Tek® O.C.T.™ Compound (Sakura, Finetek USA Inc., Torrance, CA, USA) and further stored at −80 °C until it was sectioned into ~30–40 µm-thick slices using a Leica CM1850 cryotome and placed in phosphate-buffered saline (PBS) ready for immunostaining. Both brains from wild-type mice and 13C6-lysine-pulsed mice were treated in the same way as mentioned above. Pulsing of 13C6-lysine was conducted as previously described [12 (link)]. Briefly, wild-type mice were fed with 13C6-lysine containing food for 14 or 21 days before they were sacrificed. All mouse experiments were approved by the local authority, the Lower Saxony State Office for Consumer Protection and Food Safety (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit), as also specified in the Institutional Review Board Statement.
8
Isotopic Labeling of Murine Brains
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13C6-lysine pulsing of adult mice was performed as previously described2 (link). The brains were snap-frozen immediately upon removal from the skull and stored at −80 °C. After thawing, a thick coronal slice was extracted from the center of each brain containing the hippocampal formation (~30-50 mm) and fixed in 4% PFA in PBS overnight, at 4 °C. The slices were then embedded in Tissue-Tek® O.C.T.™ Compound (Sakura, Finetek USA Inc., Torrance, CA, USA) and frozen at −80 °C. The frozen slices were sectioned into ~60 µm thick slices on a Leica CM1850 cryotome.
9
Picrosirius Red Scaffold Analysis
10
Histological Analysis of Coronary Artery
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Cardiac inter-ventricular fragments containing the left-descending coronary artery were collected from each animal group and suitably processed for microscopy histological investigations. The tissue fragments were immersed in an OCT embedding medium for 15 min at room temperature and then flash-frozen in liquid nitrogen. Cryosections were collected using the Leica CM 1850 cryotome (Wetzlar, Germany) and stained with Oil Red O and hematoxylin-eosin, as previously described [42 (link)], mounted in 90% glycerol in water, and examined using the Zeiss AXIOVert A1 microscope (Zeiss LD-Plan-Neofluar 20×/0.4 Ph2 Korr objective lens, Zeiss, Oberkochen, Germany). The histological images were captured with the Zeiss AXIOcam MRc5 Camera using ZEN imaging software (v. 2012, Blue Edition).
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