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EoE Sub Articles

All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Washing the Slides
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	<li>Prepare 10 &#181;m of unstained formalin-fixed paraffin-embedded (FFPE) sections.</li>
	<li>Place slides in a glass slide holder: use about 3-5 largest cross-sectional slides unless the tissue is minimal, and more slides are ...

deparaffinization and rehydration of tissue sections: a two-step procedure to remove paraffin wax from ffpe tissue slides followed by tissue rehydration

Source: Sugimoto, K. et al. <a href="https://www.jove.com/video/61355" target="_blank">Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the processing of FFPE or formalin-fixed paraffin-embedded sections obtained from gastrointestinal tumor tissue. The deparaffinization and rehydration steps allow the processing of the tissue specimen for subsequent analytical applications.

Deparaffinization and Rehydration of Tissue Sections: A Two-step Procedure to Remove Paraffin Wax from FFPE Tissue Slides Followed by Tissue Rehydration

Source: Sugimoto, K. et al. Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer. J. Vis. Exp. (2020)
In this video, we demonstrate the ...

Formalin-fixed paraffin-embedding or FFPE technique allows the preservation of fixed, dehydrated, and cleared tissue specimens within paraffin blocks for long-term storage. FFPE preserves proteins and other vital intracellular components without compromising the tissue morphology.
Deparaffinization, or the removal of paraffin wax surrounding the embedded tissue, is a critical step before processing any FFPE tissue specimen for downstream analysis. To begin deparaffinization, start with a glass slide carrying an unstained FFPE tissue section of interest having an appropriate thickness.
Immerse the tissue section in a xylene bath and incubate for the desired duration. Xylene, an organic solvent, dissolves the paraffin wax that has embedded and penetrated the tissue. The complete removal of paraffin wax exposes the fixed and dehydrated transparent tissue specimen.
Following deparaffinization, treat the specimen with absolute ethanol. Ethanol displaces and removes any traces of xylene from the tissue section. Subsequently, immerse the tissue in decreasing concentrations of ethanol. The process rehydrates the tissue for downstream histological analysis.
Finally, leave the slide undisturbed for the desired duration. This step allows ethanol and water to evaporate and obtain a dry rehydrated tissue section.

deparaffinization-rehydration-tissue-sections-two-step-procedure-to

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gastrointestinal Cancer

Assays and techniques

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

JoVE Journal

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Biology

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Deparaffinization and Rehydration of Tissue Sections: A Two-step Procedure to Remove Paraffin Wax from FFPE Tissue Slides Followed by Tissue Rehydration

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