High-Resolution Histology for Craniofacial Studies on Zebrafish and Other Teleost Models.

In the era of molecular biology, identification of cells and even tissues mostly relies on the presence of fluorescent tags, or of "marker gene" expression. We list a number of caveats and present a protocol for embedding, sectioning, and staining semithin plastic sections. The method is neither new nor innovative, but is meant to revive skills that tend to get lost.This easy-to-use and inexpensive protocol (1) yields high-resolution images in transmitted and polarized light, (2) can be utilized simultaneously for transmission electron microscopy, and (3) is applicable to any type of material (wild type, morphants, mutants, transgenic, or pharmacologically treated animals as well as all of their controls), provided the sample size is kept under a limit. Thus, we hope to encourage researchers to use microanatomy and histology to complement molecular studies investigating, e.g., gene function.

Three-sectioning method: A procedure for studying hard tissues and large pieces under light and electron microscopy.

The histological study of hard pieces such as tendons and calcified lesions and tissues is a field that has been gaining increased attention owing to the rapid development of implantable prostheses, among other factors. In these studies, serial sectioning is utilized to detect areas of interest throughout the entire piece, as it enables the application of the appropriate light and electron microscopy techniques in these areas. We propose the "three-sectioning method" that subjects the pieces to three consecutive cycles of embedding and sectioning to localize and study the areas of interest, as an efficient technique for these histological studies. The pieces were first embedded in epoxy resin and then cut into thick sections (approximately 300 µm) for the first cycle. Next, areas of interest selected on these thick sections were re-embedded in epoxy resin to be sectioned again (second sectioning) to obtain a series of semithin sections (1-3 µm). These semithin sections are usually studied using the most relevant techniques for light microscopy. Smaller areas of interest are selected to be cut into ultrathin sections (60-90 nm) for transmission electron microscopy. If necessary, the selected areas of the semithin sections can be embedded again, and then cut into new ultrathin sections. The different kinds of sections we have described here may also be studied using scanning electron microscopy. This systematic method facilitates correlative microscopy from lower to higher magnifications along with the usage of a broad variety of histological techniques including electron microscopy.

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy.

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM).