Ebook Pathology of the developing mouse - a systematic approach: Part 2

Part 2 book "Pathology of the developing mouse - A systematic approach" includes content: Histotechnological processing of developing mice, localization of gene expression in developing mice, protein localization in developing mice, quantitative morphological assessment in mouse developmental pathology studies, ultrastructural evaluation of developing mice,.... and other contents.
Nội dung trích xuất từ tài liệu:
Ebook Pathology of the developing mouse - a systematic approach: Part 2 9 Histotechnological Processing of Developing Mice Brad Bolon, Diane Duryea, and Julie F. Foley CONTENTS Sequence for Processing Mouse Developmental Pathology Specimens................................................. 195 Stages of Histotechnological Processing................................................................................................ 196 Fixation.............................................................................................................................................. 196 Decalcification................................................................................................................................... 199 Processing for Paraffin Sectioning..................................................................................................... 202 Paraffin Sectioning............................................................................................................................. 204 Plastic Sectioning............................................................................................................................... 205 Cryosectioning................................................................................................................................... 206 Considerations for Orienting Specimens........................................................................................... 207 Staining.............................................................................................................................................. 208 Mounting............................................................................................................................................ 209 Final Thoughts........................................................................................................................................ 209 References............................................................................................................................................... 209 A firm grasp of basic histotechnological practices is required before meaningful information may be obtained from the microscopic analysis of tissue structure. This principle is especially true for specimens harvested from developing mice. Relative to adult mice, samples from developing animals are much more liable to collapse or fragment when manipulated during histotechnological processing because of their small size and relatively high water content. Nonetheless, with regular practice researchers should be able to obtain suitable data (and publication-quality images) via microscopic examination of tissues from developing mice. The length of this practice period may be shortened considerably if the individual directly responsible for histotechnological processing is either a trained histotechnologist or is able to observe someone who is already experienced in the processing of specimens from developing mice. This chapter is designed to serve as both a basic histotechnology primer and a thorough review of the advanced methods that will be necessary for proper mouse developmental pathology analyses. Standard protocols based on the authors’ experience are presented for several common histological methods. However, these protocols will need to be optimized anew in your laboratory as variables such as reagent purity and environmental conditions (e.g., relative humidity, room temperature [RT]) may affect the end product.5 Assistance in modifying these sample protocols for your own setting may be obtained from standard histotechnology texts.1,3,8,11 Sequence for Processing Mouse Developmental Pathology Specimens Developmental pathology studies in mice commonly deal with rare events that must be discerned and characterized in small, fragile specimens. Accordingly, considerable care must be taken when plan- ning the order of histotechnological processing to ensure that artifactual damage is not introduced selectively into a specific group of samples. Care in this regard will prevent bias when interpreting the final analytical results. 195 196 Pathology of the Developing Mouse: A Systematic Approach The two major factors during tissue preparation for microscopic examination that can negatively influ- ence the study outcome are (1) the treatment of the specimens before and during histotechnological pro- cessing and (2) the sequence in which study samples are processed. All specimens—both experimental groups (e.g., genetically engineered animals, toxicant-treated animals) and corresponding controls— should be fixed for an identical period of time, preferably using fixative from the same lot. In the ideal study, experimental and control tissues should be processed, embedded, and stained together at the same time as well. Batch processing of all specimens from a single experiment in this fashion quells one major source of variation that can ruin a study. However, in practice, such simultaneity may be challenging due to the large number of consecutive sections produced when sectioning mouse embryos (especially very large near-term fetuses). If the study is so large that all samples cannot be processed in a single batch, minor adjustments to the processing sequence can reduce the likelihood that artifactual damage associated with tissue preparation will confound the final outcome. In particular, each processing batch of a large study must contain a random assortment of experimental and treated tissues. Under no circum- stances should all the controls be processed in a single batch or on a single day if experimental specimens will remain to be processed at another time. Stages of Histote ...