Ovaries were isolated 4 h after the onset of light and fixed in Bouin’s fixative at 4 °C. After dehydration, 4-μm thick plastic sections were prepared using a Technovit 8100 (Heraeus Kulzer, catalog number: RT8100) following the manufacturer’s instructions. The plastic sections were stained with hematoxylin-eosin.
Technovit 8100
Manufactured by Kulzer
Sourced in Germany
Technovit 8100 is a cold-curing resin system designed for the embedding and preparation of hard and soft tissue samples for histological analysis. It provides a durable and transparent embedding medium that allows for high-quality sectioning and staining.
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Lab products found in correlation
Axioplan 2, by Zeiss (4 mentions)
Axio imager z1 microscope, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Apotome, by Zeiss (2 mentions)
Efficient navigation zen , by Zeiss (2 mentions)
Axiovision software, by Zeiss (2 mentions)
Axio imager z2, by Zeiss (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Axiocam camera, by Zeiss (1 mentions)
Sp6 polymerase, by Promega (1 mentions)
Leica mz apo, by Leica camera (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Microm hm 355s microtome, by Thermo Fisher Scientific (1 mentions)
Isoflurane, by Viatris (1 mentions)
Entellan new, by Merck Group (1 mentions)
In vivo multispectral imaging system fx, by Kodak (1 mentions)
Rm2265, by Leica camera (1 mentions)
Stereomicroscope, by Zeiss (1 mentions)
42 protocols using technovit 8100
1
Ovarian Histological Analysis
2
Visualizing Mineralization in Undecalcified Tissue Sections
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The fixed mandibles and maxillae were dehydrated in acetone, and embedded in Technovit 8100 resin (Heraeus Kulzer GmbH, Hanau, Germany). Undecalcified sections were obtained at the thickness of 3 μm. Mineralization of the tissue was visualized by 2.5% silver nitrate staining (von Kossa stain). Bone and dentin mineralization was further assessed by following Goldner's Trichrome stain protocol (Goldner, 1938 (link)). For general morphological analysis, sections were stained with 1% toluidine blue.
3
FISH Analysis of Biofilm Architecture
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Five tooth specimens were subjected to FISH after biofilm growth to study biofilm architecture. Samples were processed as described previously [27 (link)]. In brief, specimens were fixed for 16 h in 3% paraformaldehyde and then washed three times in PBS for 20 min. Thereafter ethanol dehydration was performed in 50%, 60%, 70%, 80%, 90% and 100% ethanol-PBS (vol/vol) for one h each. Afterwards the specimens were infiltrated twice for 3 h with cold polymerizing resin (Technovit 8100; Heraeus Kulzer, Hanau, Germany). During the first 10 min of both infiltration steps, samples were placed in a vacuum chamber (Exsikkator; Kartell S.p.A. LABWARE Division, Noviglio, Italy). Specimens were then placed in polyethylene embedding molds (Flat Bottom Embedding Capsules; Electron Microscopy Sciences, Hatfield, PA, USA), and embedded overnight on ice. All procedures were performed at 4 °C. From the embedded specimens, slices of 1 mm were sectioned with a saw microtome (Ernst Leitz, Wetzlar, Germany) and decalcified in 17% EDTA for 18 days. Successful decalcification was verified by digital x-ray analysis, after which the slices were re-imbedded in resin (Technovit 8100). Finally, thin sections of <2 µm were produced on an ultramicrotome (Ultracut E; Reichert Jung Optische Werke, Wien, Austria) and mounted on coated glass slides (Polysine; Menzel-Gläser, Braunschweig, Germany).
4
Whole Mount In Situ Hybridization Protocol
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Whole mount in situ hybridization and plastic sectioning were performed as previously described [18 (link)]. The antisense riboprobes were as follows: dct and mitfa of medaka [18 (link)] and sox10 of zebrafish [30 (link)]; and sox10a, sox10b and ltk were synthesized from the plasmid described above using SP6 polymerase (Promega) after restriction enzyme digestion: sox10a and sox10b; EcoRI, ltk; XhoI (NEB). For double fluorescent in situ hybridization, the probes were labeled with digoxigenin or fluorescein, and signals were detected with anti-DIG or anti-Fluor POD-conjugated antibodies with using Tyramide Signal Amplification system (Invitrogen). For sectioning, the stained samples were embedded in Technovit 8100 (Heraeus Kulzer) and sectioned at 10μm thickness. Images of stained embryos were taken using Leica MZ APO with attached AxioCam camera (Zeiss) or Zeiss AXIOImager.M2 with attached Orca-frash 4.0 camera. Images of sections were taken using Zeiss AxioPlan2 with an AxioCam camera. Confocal images were obtained with a Zeiss LSM880 laser-scanning confocal microscope.
5
Localization of Gut Symbionts via FISH
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To localize the dominant gut symbionts, FISH was performed on 5 µm thin cross sections of the cold polymerizing resin (Technovit 8100, Heraeus Kulzer GmbH, Wehrheim, Germany) embedded gut tissue. The specificity of probes was tested and hybridization condition was achieved as described [11] (link). Shortly, the slide was hybridized with 1.5 mM of each probe (Table S1 ) in hybridization buffer containing 900 mM NaCl, 20 mM Tris-HCl (pH 8.0), 20% formamide, 1% SDS. FITC-labeled general eubacteria probe and Cy3-labeled Enterococcus-specific probe was used for detection and images were taken with an Axio Imager Z1 microscope (Carl Zeiss, Jena, Germany) [56] (link)–[58] (link).
6
Medaka Head and Pharyngeal Tissue Preparation
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The heads of medaka were fixed in a 4% paraformaldehyde solution (0.1 M phosphate buffer, pH 7.4) at 4 °C overnight, decalcified in 8% ethylenediaminetetraacetic acid (EDTA) solution at 4 °C for 3 days, then embedded in paraffin according to conventional methods. The prepared paraffin blocks were cut into sagittal and horizontal sections at a thickness of 5 µm.
Pharyngeal tissues of medaka were fixed in 4% paraformaldehyde solution (0.1 M phosphate buffer, pH 7.4) at 4 °C overnight, and then the samples were embedded in Technovit 8100 (Heraeus Kulzer, Wehrheim, Germany) with a coagulant (Heraeus Kulzer). The resin blocks were cut into horizontal sections at a thickness of 1 µm. Toluidine blue staining and tartrate-resistant acid phosphatase (TRAP) reaction were performed to identify osteoclasts [3 (link)].
Pharyngeal tissues of medaka were fixed in 4% paraformaldehyde solution (0.1 M phosphate buffer, pH 7.4) at 4 °C overnight, and then the samples were embedded in Technovit 8100 (Heraeus Kulzer, Wehrheim, Germany) with a coagulant (Heraeus Kulzer). The resin blocks were cut into horizontal sections at a thickness of 1 µm. Toluidine blue staining and tartrate-resistant acid phosphatase (TRAP) reaction were performed to identify osteoclasts [3 (link)].
7
In situ Localization of Bacterial Symbionts in Beetle Guts
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Bacterial symbionts were localized in the gut of adult beetles by designing specific diagnostic fluorescence in situ hybridization probes for each taxon (Supplementary Table 6 ). Different regions of the guts of four adult beetles were dissected under sterile water, fixed in 70% ethanol and embedded in Technovit 8100 cold-polymerizing resin (Heraeus Kulzer, Hanau, Germany) as previously described74 (link)75 (link). Sections (10 μm) were prepared with a diamond knife on a Microm HM 355S microtome (Thermo Fisher Scientific), and hybridized at 50 °C with the specific Cy3-labelled probe for each taxon, combined with a Cy5-labelled general eubacterial probe (EUB338) and 4,6-diamidino-2-phenylindole. Fluorescence images were recorded with an AxioImager Z1 microscope (Carl Zeiss AG, Oberkochen, Germany) at 400 × magnification using the z-stack option to combine different focus planes and the mosaic tool.
8
Fixation and Sectioning of Planula Larvae
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Planula larvae were fixed in Zamboni’s fixative (2% paraformaldehyde, 15% saturated picric acid, 0.15 M phosphate buffer (PB) (pH 7.3)) for 1 h in an ice bath, and then serially dehydrated and infiltrated with a plastic resin, Technovit 8100 (64709012, Heraeus Kulzer, Wehrheim, Germany) for more than 10 h at 4 °C. After hardening, samples were sectioned into 2-µm slices with glass knives. For general histology, sections were stained with 0.5% toluidine blue in 0.1 M PB (pH 7.4) for 5 min.
9
Brain Histological Preparation Protocol
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The X-ray images of brains were taken under anesthesia (Isoflurane: Mylan Inc., Canonsburg, PA, USA) using In-Vivo Multispectral Imaging System FX (Kodak, New Haven, CT, USA). The brains of 1-week old mice were fixed in 4% PFA for 2~7 days, dehydrated by EtOH and xylene, and then embedded in paraffin. The brains of adult mice were fixed in 1~2 days, dehydrated by 100% acetone, and subsequently embedded in plastic resin (Technovit 8100, Heraeus Kulzer GmbH, Hanau, Germany). The 5-μm sections were subjected to hematoxylin and eosin staining and then enclosed with Entellan New (Merck, Darmstadt, Germany).
10
Quantitative Lung Tissue Analysis
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Lung tissues were fixed and sampled as described for TEM. Tissue slices sampled for evaluation by light microscopy were embedded in glycol methacrylate (Technovit 8100®, Heraeus Kulzer) to minimize tissue deformation74 (link). A coherent test system consisting of test lines and test points was used to determine the surface density of the area of the alveolar epithelium as well as the volume fraction of the parenchymatous tissue within lung parenchyma75 (link). The arithmetic mean thickness of septal walls was calculated as a volume-to-surface ratio of parenchymatous tissue76 (link).
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