Assessment of various whole organism cryosectioning protocols for further optimization
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Citation
Abstract
Cryosectioning is the practice of rapidly freezing a tissue sample and, using a cryostat, to section the material at very thin increments in order to visualize the morphology or presence of fluorescent proteins at a high degree of resolution. There are distinct advantages to the use of cryosectioning over other sectioning modalities including speed of processing from beginning to end, preservation of antigenicity of samples and fewer steps required. Current cryosectioning techniques make visualization of sample morphology difficult when sectioning a whole organism. This is because the distinct characteristics of different tissue types can result in excess artifact and tissue distortion when sectioned. Lipids tend to smudge, calcified tissue tends to splinter, and larger radius organisms don’t freeze uniformly resulting in ice crystallization and cellular lysing. If these issues can be overcome, whole organism cryosectioning would allow for better visualization of organ interfaces and more accurate morphology that is not damaged during dissection. OBJECTIVES: It is the objective of the paper to outline five different whole organism cryosectioning preparation protocols and determine which protocol is most appropriate for further optimization.
METHODS: Five protocols were compared, with two mice undergoing each protocol. Each protocol will have a euthanasia and perfusion step, a decalcification step, a delipidation step, a wash step, a freezing step, and a sectioning/mounting step. Additionally, some involve a dehydration step. While each protocol follows a similar pattern, the procedures for each step vary from one protocol to another. At the conclusion, each mouse will be sectioned, and sections analyzed for quality.
RESULTS: The protocol that yielded the highest quality sections was Mouse 2 protocol. On average, Mouse 2 yielded sections that were 25.0 % higher quality than the next highest. Protocols for Mice 1, 3, 4, 5 and control yielded lower quality results.
CONCLUSION: The results indicate that Mouse 2 protocol is the best suited for future optimization, while the protocols used on mice 1, 3, 4 & 5 were determined to be of lower quality. There are a number of possible explanations for why these protocols yielded such poor sections including protocol design, temperature differences between the block and blade during sectioning, tissue adherence and more.
Description
2024