To distinguish between calcification and ossification of the mineralized parts, one larynx (high line male #25) was embedded in epoxy resin for sectioning and histological analysis. The specimen was first fixed as described in the clearing and staining section. It was then decalcified with 20% EDTA and dehydrated with acidified dimethoxypropane prior to embedding into Agar LVR resin (Agar Scientific, Stansted, UK) using acetone as an intermediate. Sections of cured resin blocks were sliced at 1 µm section thickness with a Leica UC6 ultramicrotome equipped with a diamond knife (Diatome, Nidau, Switzerland) and analyzed with a Nikon NiU compound microscope.
Agar lvr resin
Manufactured by Agar Scientific
Sourced in United Kingdom
Agar LVR resin is a low viscosity epoxy resin designed for embedding and embedding of biological samples. It is a two-component system that cures at room temperature.
Automatically generated - may contain errors
Lab products found in correlation
Niu compound microscope, by Nikon (4 mentions)
Diamond knife, by Electron Microscopy Sciences (2 mentions)
Uc6 ultramicrotome, by Leica (2 mentions)
Ds ri2 microscope camera, by Nikon (2 mentions)
Histo jumbo diamond knife, by Electron Microscopy Sciences (2 mentions)
Ds ri2 camera, by Nikon (1 mentions)
Eclipse e800, by Nikon (1 mentions)
5 protocols using agar lvr resin
1
Histological Analysis of Larynx Mineralization
2
Ultrastructural Analysis of Biological Specimens
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The examined samples were collected as given in detail in the original description by Grischenko et al. (2018 ). Preparations for scanning electron microscopy were also conducted as mentioned by Grischenko et al. (2018 ). Samples were analyzed and documented with a Nikon SMZ25 stereomicroscope equipped with a Nikon DsRi2 camera (Nikon). Three specimens were dehydrated with acidified dimethoxypropane followed by infiltration into Agar LVR resin (Agar Scientific) via acetone. Cured resin blocks were serially sectioned with a Leica UC6 ultramicrotome (Leica Microsystems). Sections were stained with toluidine blue, sealed in resin, and documented with a Nikon NiU compound microscope with a Nikon DsRi2 camera. The resulting image stacks were edited with FIJI (Schindelin et al., 2012 ) before importing them into the visualization software Amira (ThermoFisher). Further processing included section registration, segmentation, and visualization of segmentations as surface models and surrounding tissues as volume rendering.
3
Tissue Preparation for Microscopy
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For sectioning, samples were post-fixed in ~ 1% aqueous osmium tetroxide for one hour at room temperature followed by several rinses in distilled water. Afterwards, samples were decalcified in 2–4% ascorbic acid for two to three weeks with daily changes. Dehydration was conducted with acidified 2–2-dimethoxypropane and acetone was used as intermediate to transfer dehydrated specimens into Agar LVR resin (Agar Scientific, Stansted, Essex, UK). Ribbons of serial semithin sections of 1 μm thickness were conducted with a Diatome HistoJumbo diamond knife (Diatome, Biel, Switzerland) on a Leica UC6 ultramicrotome (Leica Microsytems, Wetzlar, Germany) as described by Ruthensteiner [34 (link)]. Staining was performed with toluidine blue for a few seconds at 60 °C. Sections were photographed with a Nikon Eclipse E800 with a Nikon DsFi3-U3 microscope camera or a Nikon NiU with a DsRi2 microscope camera (Nikon, Tokyo, Japan).
4
Histological Analysis of Beetle Neuroanatomy
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Heads of D. bosnicus and D. discolor were cut from the remaining body for histological processing. First samples were dehydrated with acidified dimethoxypropane followed by three rinses with acetone before being infiltrated and embedded in Agar LVR resin (Agar Scientific, Stansted, UK). Cure resin blocks were serially sectioned with a Diatome HistoJumbo diamond knife (Diatome, Nidau, Switzerland) at 1 µm section thickness on a Leica UC6 ultramicrotome (Leica microsystems, Wetzlar Germany). Sections were stained with 1% toluidine blue and sealed in epoxy resin. Analysis and photography of the serial sections was conducted on Nikon NiU compound microscope with a Nikon DsRi2 microscope camera (Nikon, Tokyo, Japan).
Image stacks were converted to greyscale and contrast-enhanced with F:IJI28 (link) and subsequently imported into the visualization software (Thermo Fisher Scientific). Alignment of consecutive sections was conducted with the AlignSlices Tool of Amira. Structures of interest (tentorium, nervous system and digestive tract) were semi-manually reconstructed by labelling with a brush and interpolating several consecutive sections. Surfaces were calculated from the segmentation masks, followed by surface optimization using iterated smoothing and polygon-reduction steps. Snapshots were taken with the Amira software.
Image stacks were converted to greyscale and contrast-enhanced with F:IJI28 (link) and subsequently imported into the visualization software (Thermo Fisher Scientific). Alignment of consecutive sections was conducted with the AlignSlices Tool of Amira. Structures of interest (tentorium, nervous system and digestive tract) were semi-manually reconstructed by labelling with a brush and interpolating several consecutive sections. Surfaces were calculated from the segmentation masks, followed by surface optimization using iterated smoothing and polygon-reduction steps. Snapshots were taken with the Amira software.
5
Histological Analysis of Larynx Mineralization
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