The Experts below are selected from a list of 3138 Experts worldwide ranked by ideXlab platform
Hui Gong - One of the best experts on this subject based on the ideXlab platform.
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Paraffin Embedding for large volume bio tissue
Scientific Reports, 2020Co-Authors: Ouyang Zhanmu, Hui Gong, Xiaoying YangAbstract:Acquiring ultrahigh-resolution three-dimensional images of large-volume tissues non-human primate tissues was an enormous challenge. Given the preservation of structure and excellent sectioning property, formalin-fixed Paraffin-Embedding method had an enormous potential for three-dimensional reconstruction of fine structures, based on the very thin histological sections and optical images. However, maintaining the structure uniformly in large-volume tissues was difficult during the complex processes. In this study, we presented a detailed protocol for the whole mouse, rat, rabbit brains, and even for the macaque hemisphere. The entire protocol took about 2–30 days to complete for a large sample, including fixation, dehydration, clearing, wax immersion and Embedding. In addition, it could be applied to other species and organs, while the Embedding processes depended on the size and the type of organs. This method had wide applicability to serve as a baseline for further technique development.
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maintenance of fluorescence during Paraffin Embedding of fluorescent protein labeled specimens
Frontiers in Neuroscience, 2019Co-Authors: Ouyang Zhanmu, Peilin Zhao, Yang Yang, Xiaoquan Yang, Hui GongAbstract:Paraffin Embedding is widely used in microscopic imaging for preparing biological specimens. However, owing to significant fluorescence quenching during the Embedding process, it is not compatible with fluorescent-labeling techniques, such as transgenic and viral labeling using green fluorescent protein (GFP). Here, we investigate the quenching mechanism and optimize the Embedding process to improve the preservation of fluorescence intensity. The results show that dehydration is the main reason for fluorescence quenching during Paraffin Embedding, caused by the full denaturation of GFP molecules in ethyl alcohol. To evaluate fluorescent and morphological preservation, we modified the Embedding process using tertiary butanol (TBA) instead of ethyl alcohol. Fluorescence intensity following TBA dehydration increased 12.08-fold of that observed in the traditional method. We obtained uniform fluorescence maintenance throughout the whole mouse brain, while the continuous apical dendrites, spines, and axon terminals were shown evenly within the cortex, hippocampus, and the amygdala. Moreover, we embedded a whole rat brain labeled with AAV in the prelimbic cortex (Prl). With the axon terminals in different areas, such as the caudate putamen, thalamus, and pyramidal tract, the results showed a continuous tract of Prl neurons throughout the whole brain. This method was also suitable for tdTomota labeled samples. These findings indicate that this modified Embedding method could be compatible with GFP and provides a potential turning point for applications in the fluorescent labeling of samples.
Yuichi Sato - One of the best experts on this subject based on the ideXlab platform.
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the amex method a multipurpose tissue processing and Paraffin Embedding method extraction of protein and application to immunoblotting
American Journal of Pathology, 1992Co-Authors: Yuichi Sato, Kiyoshi Mukai, Shuichiroh Furuya, Toru Kameya, Setsuo HirohashiAbstract:Abstract The authors have previously reported a new fixation and Paraffin-Embedding method (the AMeX method), which preserves many antigens as well as high molecular-weight DNA and RNA that are normally destroyed by the routine formalin fixation and Paraffin-Embedding process. In the present study, the authors analyzed the preservation of protein suitable for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting in tissue fixed by the AMeX method. The method used for extraction of protein from AMeX-processed tissue sections after deParaffinization was the same as that for extraction from fresh tissues. The total amount of protein extracted from 50-mg (wet weight) AMeX-processed mouse liver tissue was the same as that from fresh tissue. The electrophoretic mobility and staining intensity of protein on SDS-polyacrylamide gel, and the immunoblotting pattern and staining intensity with several antibodies, were identical for both AMeX-processed and fresh tissue. Degradation of protein was minimal for storage periods of 2 years in Paraffin block. The authors also showed that pellets of cultured cells can be processed by this method for immunologic analysis. This new fixation and Paraffin-Embedding method is a useful tool for obtaining information on correlations between morphologic features and immunochemical and molecular biological data.
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the amex method a multipurpose tissue processing and Paraffin Embedding method iii extraction and purification of rna and application to slot blot hybridization analysis
The Journal of Pathology, 1991Co-Authors: Yuichi Sato, Kiyoshi Mukai, Shuichiroh Furuya, Yukio ShimosatoAbstract:: RNA was extracted from tissues processed by a new fixation and Paraffin-Embedding method (the AMeX method) and examined by Northern blot analysis and slot-blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deParaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern blot hybridization analysis. The intensity of ethidium bromide staining and the hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by slot-blot hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.
Yukio Shimosato - One of the best experts on this subject based on the ideXlab platform.
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the amex method a multipurpose tissue processing and Paraffin Embedding method iii extraction and purification of rna and application to slot blot hybridization analysis
The Journal of Pathology, 1991Co-Authors: Yuichi Sato, Kiyoshi Mukai, Shuichiroh Furuya, Yukio ShimosatoAbstract:: RNA was extracted from tissues processed by a new fixation and Paraffin-Embedding method (the AMeX method) and examined by Northern blot analysis and slot-blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deParaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern blot hybridization analysis. The intensity of ethidium bromide staining and the hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by slot-blot hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.
Giacomina Rossi - One of the best experts on this subject based on the ideXlab platform.
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Effects of formalin fixation, Paraffin Embedding, and time of storage on DNA preservation in brain tissue: A brainnet Europe study
Brain Pathology, 2007Co-Authors: Inés Ferrer, Tim Ströbel, Jeanne Bell, Judith Armstrong, Sabina Capellari, Piero Parchi, Herbert Budka, Giuseppe Giaccone, Thomas Arzberger, Giacomina RossiAbstract:There is a large amount of tissue stored in brain collections and brain banks, but little is known about whether formalin-fixed tissues and Paraffin blocks stored for years in brain banks are suitable for the retrospective genetic studies. The study was carried out in order to: (i) compare DNA preservation in frozen, formalin-fixed and Paraffin-embedded tissues stored for different periods; (ii) study point mutations and triplet expansions in frozen, formalin-fixed and Paraffin-embedded material stored for variable periods, and using different fixative solutions; (iii) compare different methods to optimize DNA extraction and DNA amplification from suboptimally preserved brain tissue. DNA preservation is suitable for genetic studies in samples stored at -80 degrees C for several years. Formalin-fixed, Paraffin-embedded tissue was inferior to frozen tissue, but did yield adequate results in many cases depending on the type of fixative solution and time of fixation before Embedding. Prolonged fixation in formalin rarely yielded useful DNA. Similar results were obtained in samples from prion diseases. The best results were obtained by using the Qiagen kits (QIAmp DNA Micro) in frozen material, Paraffin blocks and formalin-fixed tissue. Genomiphi and TaKaRa Ex Taq methods were also assayed in Paraffin blocks and in formalin-fixed samples with limited success.
Christos Bikis - One of the best experts on this subject based on the ideXlab platform.
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virtual histology of an entire mouse brain from formalin fixation to Paraffin Embedding part 1 data acquisition anatomical feature segmentation tracking global volume and density changes
Journal of Neuroscience Methods, 2021Co-Authors: Griffin Rodgers, Georg Schulz, Alexandra Migga, Willy Kuo, Mario Scheel, Christos BikisAbstract:Abstract Background: Micrometer-resolution neuroimaging with gold-standard conventional histology requires tissue fixation and Embedding. The exchange of solvents for the creation of sectionable Paraffin blocks modifies tissue density and generates non-uniform brain shrinkage. New method: We employed synchrotron radiation-based X-ray microtomography for slicing- and label-free virtual histology of the mouse brain at different stages of the standard preparation protocol from formalin fixation via ascending ethanol solutions and xylene to Paraffin Embedding. Segmentation of anatomical regions allowed us to quantify non-uniform tissue shrinkage. Global and local changes in X-ray absorption gave insight into contrast enhancement for virtual histology. Results: The volume of the entire mouse brain was 60%, 56%, and 40% of that in formalin for, respectively, 100% ethanol, xylene, and Paraffin. The volume changes of anatomical regions such as the hippocampus, anterior commissure, and ventricles differ from the global volume change. X-ray absorption of the full brain decreased, while local absorption differences increased, resulting in enhanced contrast for virtual histology. These trends were also observed with laboratory microtomography measurements. Comparison with existing methods: Microtomography provided sub-10 μm spatial resolution with sufficient density resolution to resolve anatomical structures at each step of the Embedding protocol. The spatial resolution of conventional computed tomography and magnetic resonance microscopy is an order of magnitude lower and both do not match the contrast of microtomography over the entire Embedding protocol. Unlike feature-to-feature or total volume measurements, our approach allows for calculation of volume change based on segmentation. Conclusion: We present isotropic micrometer-resolution imaging to quantify morphology and composition changes in a mouse brain during the standard histological preparation. The proposed method can be employed to identify the most appropriate Embedding medium for anatomical feature visualization, to reveal the basis for the dramatic X-ray contrast enhancement observed in numerous embedded tissues, and to quantify morphological changes during tissue fixation and Embedding.
