Mice were anaesthetised using Isoflurane. Their right atrium was incised and PBS (Invitrogen, Manchester, UK) was injected into the left ventricle. The brain tissue was stored in 4% PFA (Sigma-Aldrich) for 24 hours. The skull was removed and the brain tissue was fixed in 4% PFA for another 24 hours. The tissues were stored in 30% sucrose (Sigma-Aldrich) at 4 °C, until sectioned using a microtome (Carl Zeiss, Welwyn Garden City, UK) to generate 40 µm transverse sections.
Microtome
Manufactured by Zeiss
Sourced in Germany, Switzerland
The Microtome is a precision instrument used to cut extremely thin sections of materials for microscopic examination. It is designed to produce uniform, high-quality samples from a variety of materials such as biological tissues, polymers, and metals. The Microtome utilizes a sharp blade to slice the sample into thin slices, allowing for detailed analysis and observation under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Nbt bcip precipitation, by Roche (3 mentions)
Anti dig alkaline phosphatase antibody, by Roche (3 mentions)
Axiophot microscope, by Zeiss (2 mentions)
Nis elements br 3, by National Instruments (2 mentions)
Eclipse ti u microscope, by Nikon (2 mentions)
Axio imager m2, by Zeiss (2 mentions)
Blocking reagent, by Roche (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Entellan, by Merck Group (1 mentions)
Mayer s hemalaun solution, by Carl Roth (1 mentions)
Micromanipulator, by Leica (1 mentions)
M2 medium, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
M16 medium, by Merck Group (1 mentions)
Fluoro ruby, by Merck Group (1 mentions)
Color view 1 camera, by Olympus (1 mentions)
Roti histofix, by Carl Roth (1 mentions)
Mirax scanner, by Zeiss (1 mentions)
21 protocols using microtome
1
Brain Tissue Fixation and Sectioning
2
Histological Analysis of Caecum Morphology
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After careful dissection and fixation, tissues were embedded in paraffin. Serial sections of five μm were cut using a microtome (Carl Zeiss AG, Feldbach, Switzerland) and stained with haematoxylin-eosin (HE). The histological changes in the caecum were quantified in a blinded manner by two investigators (range 1–24) for morphological features (villous stunting, villous epithelial injury and crypt distortion) and infiltration of immune cells (intraepithelial lymphocytes and infiltrating lymphocytes and plasma cells in the LP) as outlined in Supplementary Table 3 .
3
Histopathological Analysis of Lung Tissues
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After BAL harvesting, either the whole lung or the left lobe was inflated by 5 ml of 10% phosphate buffered formaldehyde solution (PFA) (AppliChem) and then gently removed and immersed in 10% PFA. An average of three mice per group at each experimental time point was used for histopathological analysis. After fixation for 24 h, dissected lung tissues were dehydrated through a series of solutions with increasing concentrations of ethanol and subsequently embedded in paraffin blocks. 3 µm thick adjacent sections were cut by the microtome (Carl Zeiss), so that all parts of the samples were represented on the slides. Prior to H&E staining, slides were baked at 60°C for 30 min. Subsequently, lung sections were prepared for histopathological staining by deparaffinization in xylene, and rehydration in a decreasing ethanol series (100, 90, 80, and 70%, respectively) and distilled water. Slides were then stained with H&E according to the manufacturer’s protocols to determine histopathological changes and fibrosis. Briefly, lung sections were incubated in Mayer’s Hemalaun solution (Carl Roth) for 8 min, rinsed quickly in 0.3% acid–alcohol solution, washed, and then transferred into 0.5% eosin G solution (Carl Roth) for 8 min. Sections were washed in tap water and dehydrated in a graded ethanol series and covered with Entellan (Millipore).
4
Visualizing Dnmt1 Isoforms in Mouse Zygotes
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Mouse zygotes were collected from superovulated BCF1 (C57BL/6 × CBA/CA) females as described previously (28 ). Briefly, female BCF1 mice at 5 weeks of age were injected with 5 IU of pregnant mare serum gonadotrophin (PMSF), followed by 5 IU of human chorionic gonadotropins (hCG) 48 h apart, and mated with male mice. Successful mating was determined the following morning by detection of a vaginal plug. Mouse zygotes were transferred to M2 medium (Sigma) containing 0.1% (w/v) hyaluronidase to remove cumulus cells and cultured in M16 medium (Sigma) at 37°C, 5% CO2 in air (29 (link)).
Microinjection was performed 24 h after human chorionic gonadotropin injection (30 (link)). To visualize the pronuclei, cumulus-free zygotes were centrifuged at 13,000 rpm for 5 min. Linearized GFP-Dnmt1sc− or GFP-Dnmt1oc− expression construct (4–20 ng/μl) was microinjected into the male pronuclei of mouse zygotes using an inverted microscope equipped with a micromanipulator (Leica). The injected zygotes were then cultured in M16 media for 48 h. The resulting four- or eight-cell stage embryos were collected, fixed using 4% formaldehyde and mounted with DAPI (Vector). The localization of Dnmt1 isoforms was evaluated by detecting GFP signals under a fluorescence microscope equipped with a microtome (Zeiss).
Microinjection was performed 24 h after human chorionic gonadotropin injection (30 (link)). To visualize the pronuclei, cumulus-free zygotes were centrifuged at 13,000 rpm for 5 min. Linearized GFP-Dnmt1sc− or GFP-Dnmt1oc− expression construct (4–20 ng/μl) was microinjected into the male pronuclei of mouse zygotes using an inverted microscope equipped with a micromanipulator (Leica). The injected zygotes were then cultured in M16 media for 48 h. The resulting four- or eight-cell stage embryos were collected, fixed using 4% formaldehyde and mounted with DAPI (Vector). The localization of Dnmt1 isoforms was evaluated by detecting GFP signals under a fluorescence microscope equipped with a microtome (Zeiss).
5
Histological Analysis of Mouse Ear Thickness
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The ears of the mice were treated as described above. Subsequently, they were cut and fixed in 4% PFA for 24 h. After three washes in PBS, they were embedded in paraffin and cut into sections on a microtome (Zeiss). The sections were stained with hematoxylin and eosin. Images were taken using an Axiophot microscope (Zeiss) with a magnification of 125×. For its quantification, the thickness of the ear was measured using ImageJ imaging software (NIH) and expressed as mean ± standard error of the mean (S.E.M.). Student’s t-test was applied for the statistical analysis, and a value of p < 0.05 was considered statistically significant.
6
In Vivo Axonal Tracing Utilizing Fluoro-Ruby
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Fluoro-Ruby is a fluorescent rhodamine-conjugated dextran, widely used in literature, and already previously used by our group, to study in vivo axonal transport within the central nervous system (CNS) [13 (link),42 (link)]. The preparation of the Fluoro-Ruby (Millipore) solution was done as previously described [13 (link)]. Fluo-Ruby was administered with an intraspinal injection at the level of the dorsal funiculus rostral to the lesion epicentre, at T6/T7 using a 5 µL Hamilton microsyringe 25 days after treatment. Injection volumes ranged from 0.5–1 µL and the injections were gradually performed over a period of 10–15 minutes [13 (link)]. The animals were then allowed to recover and perfused 10 days after the tracer injection. The perfusion was done with neutral buffered formaldehyde as previously described. The spinal cord was then removed, post-fixed overnight in the same fixative solution, and then included in OCT (Killik Bio-Optica, Milano, Italy). The cords were then sectioned by means of a microtome (Zeiss, Oberkochen, Germany) cutting sections 18 µm in thickness. This analysis was performed on 3 animals per experimental group: 1) lesioned mice, 2) lesioned mice transplanted with LS, and 3) lesioned mice transplanted with MALS.
7
Liver Histology and Morphology Analysis
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To evaluate liver morphology, each liver was fixed in 10% formaldehyde solution for 8 h, in individual cassettes. Subsequently, the fixed samples were kept overnight in 70% alcohol. The samples were then dehydrated through a series of baths in 95% alcohol, 100% alcohol, and xylene. Once the samples were dehydrated, the tissue samples were embedded in paraffin at 60 °C. A microtome (Zeiss, Jena, Germany) was used to cut the samples into 5-micron slices. The slices were stained with hematoxylin and eosin (H&E), the cellular morphology was evaluated, and the intracellular vacuoles were quantified. Twenty images from each animal were obtained using a Nikon Eclipse Ti-U microscope at 20× magnification coupled with a Nikon DS-R1 digital camera and NIS-Elements BR 3.1 software. These images were projected onto a high-resolution LCD monitor.
8
Histological Analysis of Femoral Bone
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The femurs were decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for 3 weeks, embedded in paraffin, and then sectioned using a microtome (5 µm-thick; Carl Zeiss AG). Tissue sections were mounted on a slide dryer for 1 day. H&E staining was performed to measure the trabecular area. The trabecular areas were captured in H&E-stained sections under a light microscope (Olympus Corporation, magnification, ×100) and its was measured with ImageJ version 1.46. To confirm the bone resorption and bone formation parameters in femoral tissues, we stained them using the TRAP and masson-goldner’s trichrome staining kit according to the manufacturer’s protocol. The stained tissues were captured under a light microscope (Olympus Corporation, magnification, ×100 and ×200). Bone resorption parameters such as osteoclast surface per bone surface (Oc.S/BS) and osteoclast number per bone surface (Oc.N/BS) and bone formation parameters such as Osteoblast surface per bone surface (Ob.S/BS), the osteoblast number per bone surface (Ob.N/BS) were measured with ImageJ version 1.46.
9
Histological Evaluation of Inflammatory Cells
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For histological examination, 3 samples of the full-thickness wound and 1 sample of the contralateral healthy abdominal wall were collected from each animal. Each sample was 0.5 cm thick. The samples were fixed in 4% neutral buffered formalin for 48 hours, dehydrated with gradient alcohol series, cleared in xylene and eventually embedded in paraffin. Serial sections (8 μm) were obtained using a Leitz (Wetzlar, Germany) microtome, stained with hematoxylin and eosin (H&E) and examined with a Zeiss (Oberkochen, Germany) Axiophot microscope.
We evaluated the presence of inflammatory cells: neutrophils, eosinophils, lymphocytes, macrophages and mast cells, in pericatheter blank tissues. A total of 15 sections were evaluated for statistical analysis (4 sections per group – corresponding to a different animal of each experimental group plus 1 contralateral healthy sample per group). For each section, at least 8 randomly determined areas of connective tissue were examined at 20× magnification and 100 cells per area were counted. The rate of inflammatory cells was assessed using a semiquantitative scale, with scores ranging from 0 to 4 by 3 independent observers (Table 1 ). Histological scores are reported as median and 25–75 percentile (interquartile range, IQR).
We evaluated the presence of inflammatory cells: neutrophils, eosinophils, lymphocytes, macrophages and mast cells, in pericatheter blank tissues. A total of 15 sections were evaluated for statistical analysis (4 sections per group – corresponding to a different animal of each experimental group plus 1 contralateral healthy sample per group). For each section, at least 8 randomly determined areas of connective tissue were examined at 20× magnification and 100 cells per area were counted. The rate of inflammatory cells was assessed using a semiquantitative scale, with scores ranging from 0 to 4 by 3 independent observers (
10
Histological Analysis of Osteoporotic Femurs
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Osteoporotic femurs and fractured femurs were fixed in neutral buffered formalin (NBF) and subsequently washed in running water for 24 h. Following this, they underwent decalcification with ethylenediaminetetracetic acid disodium (EDTA) for a period of 3 weeks. The samples were then degreased and dehydrated using ethanol and finally embedded in paraffin. Tissue sections of 5 μm thickness were obtained using a microtome (Carl Zeiss AG). The sections were then stained with hematoxylin and eosin (H&E), and the stained tissues were observed under a 100-fold magnification using an optical microscope. For analysis, the bone trabecular area was used as the standard, which included the area below the growth plate in the proximal femur, with the horizontal length being 2/3 of the growth plate length. In the case of the fractured femur, the fracture line was aligned at the center of the photograph. The bone density within the femur was measured using the image analysis program Image J, Ver. 1.46 (National Institutes of Health).
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