Each liver was fixed in 10% formalin solution for 36 h at 37°C, embedded in paraffin, sectioned into 4 µm-thick slices and stained with hematoxylin for 10 min at 37°C. Stained sections were imaged using a light microscope (Leica DM200; Leica Microsystems Ltd.) using a 40X objective lens and a color camera.
Dm200
Manufactured by Leica
Sourced in Germany, Austria
The Leica DM200 is a high-performance microscope designed for routine laboratory applications. It features a stable and vibration-resistant stand, providing a reliable platform for precise observations. The microscope is equipped with a range of objective lenses, allowing for a variety of magnification levels to accommodate different sample types and research needs.
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8 protocols using dm200
1
Histological Analysis of Liver Tissue
2
Microstructural Characterization of Materials
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The specimens were grounded, polished, and ultrasonically cleaned with ethanol. Then, the microstructures were investigated using SEM. Moreover, the crystalline structure was observed using an optical microscope (Leica DM200, Leica Microsystems, Germany) after etching with a nitric acid alcohol solution (4%). Furthermore, the phase compositions were identified using X-ray diffraction (XRD, D8 Advance diffractometer, Bruker Inc., German) at a scanning rate of 8 min−1.
3
Transmission Electron Microscopy of Animal Eyes
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Three animals per group (12 months-old) were used. Eyes from wild type, DE8 and D27 were enucleated, the cornea was perforated using a needle to create a small hole and eyes were immersed in fixative solution (2.5% glutaraldehyde, 2% PFA in 0.1 M phosphate buffer) incubation at 4°C overnight. After several rinses (0.1 M phosphate buffer) using a shaker, eyes were post-fixed in 1% osmium tetroxide and 0,8% K4Fe(CN)6 in the dark for 2h at 4ºC temperature, rinsed in double distilled water to remove the osmium. Eyes were dehydrated in ascending concentrations of acetone, and embedded in Epon (EMS). Blocs were obtained after polymerization at 60ºC for 48 h. Semithin sections of 1 µm in thickness were obtained using a UC6 ultramicrotome (Leica Microsystems, Vienna, Austria), dyed with 0.5% methylene blue and observed in an optic microscope Leica DM200 (Leica Microsystems, Vienna, Austria). Ultrathin sections of 60 nm thick were obtained using a UC6 ultramicrotome (Leica Microsystems, Austria), and stained with 2% uranyless and lead citrate. Sections were observed in a Jeol EM J1010 (Jeol, Japan), and images were acquired at 80 kV with a 1k x 1k CCD Megaview camera.
4
Alginate-Carbohydrate Films Ultrastructure
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Alginate-carbohydrate films were fixed in a 2.5% glutaraldehyde in cacodylate buffer 0.1M for 90 min. To prevent the films from dissolving in the solution, 5% calcium chloride solution was added to the fixative solution. Films were rinsed four times with 5% CaCl2 solution and once with milliQ water and postfixed with 1% osmium tetroxide overnight. The films were then rinsed with 0.1M cacodylate buffer before being dehydrated in an ethanol series (50%, 70%, 90%, 96% and 100%, for 10 min each).
They were infiltrated and embedded in Spurr's low-viscosity resin (EMS, Hatfield, USA). Sections of 500 nm in thickness were obtained using a UC6 ultramicrotome (Leica Microsystems, Vienna, Austria), dyed with 0.5% methylene blue and observed in an optic microscope Leica DM200 (Leica Microsystems, Vienna, Austria).
Sections of 60 nm in thickness were obtained using a UC6 ultramicrotome (Leica Microsystems, Vienna, Austria) and stained with 2% uranyl acetate and lead citrate.
Sections were observed in a Tecnai Spirit microscope (EM) (FEI, Eindhoven, The Netherlands) equipped with a LaB6 cathode. Images were acquired at 120 kV with a 1376 x 1024 pixel CCD Megaview camera.
They were infiltrated and embedded in Spurr's low-viscosity resin (EMS, Hatfield, USA). Sections of 500 nm in thickness were obtained using a UC6 ultramicrotome (Leica Microsystems, Vienna, Austria), dyed with 0.5% methylene blue and observed in an optic microscope Leica DM200 (Leica Microsystems, Vienna, Austria).
Sections of 60 nm in thickness were obtained using a UC6 ultramicrotome (Leica Microsystems, Vienna, Austria) and stained with 2% uranyl acetate and lead citrate.
Sections were observed in a Tecnai Spirit microscope (EM) (FEI, Eindhoven, The Netherlands) equipped with a LaB6 cathode. Images were acquired at 120 kV with a 1376 x 1024 pixel CCD Megaview camera.
5
Quantifying Aortic Atherosclerosis in Mice
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Mice were anesthetized, and the mouse hearts with aortic roots as well as whole aortas were harvested. For inner face plaque analyses, the whole aortas without adipose tissue, fixed in 4% paraformaldehyde overnight, were stained with Oil Red O solution for 15 min and washed with 70% ethanol. For aortic root cross-section analysis, the mouse hearts with aortic roots were embedded and sliced into several sections, followed by staining with Oil Red O solution as described in our previous study [28 (link)]. Images were viewed by a biomicroscope (DM200, Leica) [47 (link), 48 (link)]. The percentage of Oil Red O-positive areas, presumed to be atherosclerotic lesions, was quantified by ImageJ software.
6
Histological Assessment of Bone Remodeling
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Tibias from all four groups were fixed overnight in 4% Paraformaldehyde, embedded in methyl methacrylate, sectioned (7-μm thickness), and processed for von Kossa and van Gieson (VKVG) staining. Stained bone sections were analysed for osteoid volume/bone volume (OV/BV) using the Osteomeasure software (Osteometrics, Inc.) at objective 40. The region of interest (ROI) was selected within the fracture healing site (5 random locations for each sample). The sections were also decalcified in a Cal-Ex decalcifier solution (Fisher, Pittsburgh, PA) overnight and processed for Alcian Blue and van Gieson (ABVG) staining. Images were taken at room temperature using a light microscope (DM200; Leica) with a 5, 20 or 40-objective. All histological images were captured using a camera (DP72; Olympus), acquired with DP2-BSW software (XV3.0; Olympus), and processed using Photoshop (Adobe).
7
Histological Analysis of Excision Wound Healing
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Histological studies were performed using the excision wound model by collecting the wound tissues on the 14th postoperative day followed by blotting, drying, and fixing using formalin (10%). The tissues were dehydrated using acetone and then embedded in paraffin wax. Tissue was sectioned using a microtome keeping a thickness of 4–8 μm and were stained using hematoxylin-eosin (1:1) followed by their observation under the microscope (Leica DM-200) for histological examinations.20 (link)
8
Calcein Double Labeling for Bone Histomorphometry
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For calcein double labeling, 5-week-old mice were injected intraperitoneally with 10 μl/g body weight of calcein solution (0.25% calcein and 1% NaHCO3 dissolved in 0.15 M NaCl) twice at a 3-day interval. Mice were euthanized 4 days after the second injection and skeletal tissues were processed for histomorphometric analyses. For plastic sectioning, vertebrae and long bones were fixed overnight in 4% paraformaldehyde/phosphate-buffered saline (PBS), embedded in methyl methacrylate, and sectioned (7-μm thickness). Von Kossa and van Gieson (VKVG), Von Kossa and Safranin O (VKSO), Toluidine blue or TRAP staining were applied. Stained bone sections were analyzed for bone volume/tissue volume (BV/TV), osteoblast count, osteoclast count, MAR and BFR/BS analyses using the Osteomeasure software (Osteometrics, Inc.). The region of interest (ROI) was selected in the lumbar vertebrae (L3 and L4) sections avoiding the areas adjacent to the cortical bones and the growth plates. Images were taken at room temperature using a light microscope (DM200; Leica) with a 5× (numerical aperture of 0.11), 20× (numerical aperture of 0.40) or 40× (numerical aperture of 0.65) objective. All histological images were captured using a camera (DP72; Olympus), acquired with DP2-BSW software (XV3.0; Olympus Canada Inc), and processed using Photoshop (Adobe).
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