Histology of MRO was performed as previously described (Völkner et al., 2019 (link)). Briefly, MRO were fixed in modified Karnovsky’s fixative (2% glutaraldehyde, 2% paraformaldehyde in 50 mM HEPES) overnight at 4°C (Kurth et al., 2010 (link)). Samples were washed, postfixed in 1% OsO4/PBS, washed again and dehydrated in a graded series of ethanol. Samples were infiltrated in Technovit 7100 and embedded. Sections were cut at 2 μm using a rotary microtome. Sections were stained with 1% toluidine blue/0.5% borax and imaged using the Keyence Biozero 8000 fluorescence microscope.
Biozero 8000
Manufactured by Keyence
Sourced in Japan
The Biozero 8000 is a compact and versatile microscope designed for biological and medical applications. It features a high-resolution camera and advanced imaging capabilities to capture detailed images of cells, tissues, and other biological samples. The Biozero 8000 is a standalone unit that does not require a separate computer for operation.
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5 protocols using biozero 8000
1
Histological Analysis of Melanocyte-Containing Organelles
2
Melanin Synthesis in Cultured Melanocytes
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To evualuate the function of melanin synthesis in cultured melanocytes, after the indiated treatments, cells were incubated with 0.1% (w/v) levodopa (L-dopa) (Wako) for 1 h at 37 °C in an atmosphere containing 5% (v/v) CO2. The cells were then photographed under bright-field microscopy (Biozero 8000, Keyence Co., Osaka, Japan).
3
Whole-mount immunohistochemistry and resin embedding
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Whole-mount immunolabelling and embedding into the methacrylate resin Technovit 7100 (Heraeus-Kulzer, Wehrheim, Germany) was performed as described40 (link). In brief, embryos and larvae were fixed in 4% buffered PFA followed by postfixation in Methanol/DMSO41 (link). After rehydration in a graded series of methanol and PBS, the samples were blocked in 20% normal goat serum in PBS and incubated with primary antibodies (rabbit anti-GFP: TP 401 from Torrey Pines; mouse anti-GFP: clone 3E6, Invitrogen; rabbit anti β-catenin: P14L42 (link)), followed by washes in PBS and incubation with Alexa488- and/or Alexa555-coupled secondary antibodies. After staining, the samples were postfixed in 4% PFA, followed by facultative embedding into 2% agarose/PBS to facilitate proper sample orientation later during resin embedding. Finally, samples were dehydrated in a graded series of ethanol and infiltrated and embedded in Technovit 7100. Consecutive sections (2 μm) were collected on separate slides for standard histology (1% toluidine blue / 0.5% Borax) and fluorescence (mounting with Mowiol/DABCO), respectively. This way, tissue organization of one section can be correlated to protein expression on the next section which is only 2 μm apart40 (link). Images were taken on a Keyence Biozero 8000 fluorescence microscope.
4
Histomorphometric Analysis of Bone-Implant Interface
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The specimens were fixed in 10% neutral formalin solution (Wako Chemical, Tokyo, Japan) for 48 h and then, dehydrated with ascending grades of alcohol and methyl methacrylate (MMA Technovit 7200, Heraeus Kulzer, Hanau, Wehrheim, Germany). The specimens were embedded in methyl methacrylate (Technovit 7200) and polymerized. Then, the ground section of mesiodistal direction including the implant was prepared with a cutting-grinding unit (Exakt, Apparatebau, Norderstedt, Germany) and with a micro grinding polishing unit (Le Cube, PRESI, Brié-et-Angonnes, France). The ground sections, from 70 to 80 μm thickness, were stained with 0.1% toluidine blue (TB). Histological images were taken with an optical microscope (Biozero-8000, Keyence, Osaka, Japan) and the data were processed using BZ-Analyzer software (BZ-Analyzer, Keyence). The bone-implant contact (BIC) was analyzed extending from the implant shoulder to the apical end. For the analysis of the bone area around each implant, the sum bone areas between all the threads in both mesio and distal side of the implants were defined as new bone areas (NBA) (Fig. 3).
BIC was defined as the length of bone surface in direct contact with the implant surface and calculated as percentage of bone surface in direct contact to all the threads part of implant surface. The mean value of BIC and NBA in HA and DOX groups was compared.
BIC was defined as the length of bone surface in direct contact with the implant surface and calculated as percentage of bone surface in direct contact to all the threads part of implant surface. The mean value of BIC and NBA in HA and DOX groups was compared.
5
Cell Proliferation Assay Protocol
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Cells were plated at 1 × 104 cells/mL/well in 12-well plates and grown for 72 h. Cell numbers were determined using a trypan blue dye exclusion method. For the BrdU incorporation assay, cells were incubated at the same cell density described above for 48 h. BrdU (Sigma) was added to the plates (10 μM) for 30 min; then, the cells were fixed with cold methanol for 10 min, rehydrated in PBS, and incubated for 30 min in 1.5 M HCl. After three washes with PBS, the plates were incubated with a 1:50 dilution of fluorescein isothiocyanate-conjugated anti-BrdU antibody (Roche Applied Science) for 30 min at room temperature. Finally, the cells were washed three times with PBS and incubated with 10 μg/mL propidium iodide (Sigma) in PBS for 30 min at room temperature. BrdU-positive cells were examined under a microscope (Biozero-8000; Keyence, Japan).
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