Histology of plastic embedded amphibian embryos and larvae

Abstract

Summary: Amphibians including the South African clawed frog Xenopus laevis, its close relative Xenopus tropicalis, and the Mexican axolotl (Ambystoma mexicanum) are important vertebrate models for cell biology, development, and regeneration. For the analysis of embryos and larva with altered gene expression in gainof-function or loss-of-function studies histology is increasingly important. Here, we discuss plastic or resin embedding of embryos as valuable alternatives to conventional paraffin embedding. For example, microwaveassisted tissue processing, combined with embedding in the glycol methacrylate Technovit 7100, is a fast, simple, and reliable method to obtain state-of-the-art histology with high resolution of cellular details in less than a day. Microwave-processed samples embedded in Epon 812 are also useful for transmission electron microscopy. Finally, Technovit-embedded samples are well suited for serial section analysis of embryos labeled either by whole-mount immunofluorescence, or with tracers such as GFP or fluorescent dextrans. Therefore, plastic embedding offers a versatile alternative to paraffin embedding for routine histology and immunocytochemistry of amphibian embryos. genesis 50:235-250, 2012. V V C 2011 Wiley Periodicals, Inc.

Increased Expression of Beige/Brown Adipose Markers from Host and Breast Cancer Cells Influence Xenograft Formation in Mice

“…Osmium tetroxide crosslinks lipids and prevents loss of cytoplasmic lipid during subsequent tissue dehydration. Osmium tetroxide also stains lipid droplets brown (19). Following three additional two-hour washes with buffer, the tumors were dehydrated with 70%, 90%, 95%, and 100% ethanol, and embedded in glycol methacrylate (Kulzer Technovit 7100, Electron Microscopy Sciences, Hatfield, PA) according to the manufacturer’s protocol.…”

Mesodermal origin of median fin mesenchyme and tail muscle in amphibian larvae

“…Whole-mount immunolabelling and embedding into the methacrylate resin Technovit 7100 (Heraeus-Kulzer, Wehrheim, Germany) was performed as described40. In brief, embryos and larvae were fixed in 4% buffered PFA followed by postfixation in Methanol/DMSO41.…”

“…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. Images were taken on a Keyence Biozero 8000 fluorescence microscope.…”

Axolotls with an under- or oversupply of neural crest can regulate the sizes of their dorsal root ganglia to normal levels

“…Consecutive sections (2 mm) were collected on separate slides for bright field microscopy (after staining with 1% toluidine blue/0.5% Borax) and fluorescence microscopy (following counterstaining with DAPI and mounting with Mowiol/DABCO). This way, tissue organization of one section can be correlated to protein expression on the next section which is only 2 μm apart (Kurth et al, 2012). Images were taken with a Keyence Biozero 8000 fluorescence microscope.…”

“…Whole-mount immunolabelling and embedding into the methacrylate resin Technovit 7100 (Heraeus-Kulzer, Wehrheim, Germany) was performed as described (Kurth et al, 2012). In brief, larvae and juveniles were fixed in 4% buffered PFA followed by postfixation in Methanol/DMSO (Dent et al, 1989).…”