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      Advancements in 3D spheroid imaging: Optimised cryosectioning and immunostaining techniques

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          Abstract

          This article presents a modified protocol for embedding and sectioning spheroids and organoids, which are increasingly used in research due to their ability to emulate living tissue. The modifications aim to reduce the distortion and damage of these fragile structures during the embedding and sectioning process. The new method involves using optimized embedding containers, a modified embedding protocol, and optimized temperatures for cryosectioning. A heat-induced antigen retrieval protocol was tested and found to significantly increase immunostaining intensity without compromising spheroid integrity. The combined approach allowed for the creation of thinner cryosections, leading to clearer and more detailed images. The results suggest that the modified protocol could be widely adopted to enhance the imaging of spheroids and organoids.

          • •

            Paraformaldehyde fixation of spheroids

          • •

            Antigen retrieval treatment of spheroids

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            Embedding in freezing medium and cryosectioning

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          Historical evolution of spheroids and organoids, and possibilities of use in life sciences and medicine

          Marna Sakalem, Maria De Sibio, Felipe Allan da Silva da Costa … (2021)
          Article 0 comments Cited 28 times – based on 0 reviews     Review now
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            The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments

            Krzysztof Wrzesinski, Adelina Rogowska-Wrzesinska, Rattiyaporn Kanlaya … (2014)
            Introduction Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. Results Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. Summary We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance.
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              An enhanced antigen-retrieval protocol for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues.

              Justus B. Cohen, S Syrbu (2010)
              Formalin is the most commonly used fixative for light microscopy because of its preservation of -morphological details. A major adverse effect of formalin fixation is formation of cross-linkages between epitopes (amino acid residues) and unrelated proteins by formaldehyde groups. The great majority of monoclonal and polyclonal antibodies used for immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissues necessitate unmasking antigens for antigen retrieval. There are currently two major antigen-retrieval procedures based on treatment of deparaffinized tissue sections with heat or, less commonly, with enzymatic digestion. The use of various antigen-retrieval solutions and heating sources does not allow standardization of IHC staining and minimalization of interlaboratory discrepancies. We developed a novel modified antigen-retrieval protocol for reversing the effect of -formalin fixation. The key feature of this protocol is treatment of deparaffinized tissue sections at reduced constant heat (97(o)C in a water bath) for 40 min in 25 mM Tris-HCl (pH 8.5), 1 mM EDTA, and 0.05% SDS (Tris-EDTA-SDS) buffer. Sections are then immunostained with primary and secondary antibodies conjugated with polymer-labeled Horse Radish Peroxidase. Compared to conventional antigen-retrieval procedures, this protocol more efficiently reverses the effect of formalin fixation of a wide variety of cellular antigens and in most instances decreases the use of primary antibody by 2-40 times, resulting in cost savings. Moreover, this protocol eliminates the need for using different antigen-retrieval methods in the laboratory, which reduces both time and labor for medical technologists.
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                Author and article information

                Contributors
                Adelina Rogowska-Wrzesinska
                Journal
                Journal ID (nlm-ta): MethodsX
                Journal ID (iso-abbrev): MethodsX
                Title: MethodsX
                Publisher: Elsevier
                ISSN (Electronic): 2215-0161
                Publication date PMC-release: 06 October 2023
                Publication date Collection: December 2023
                Publication date (Electronic): 06 October 2023
                Volume: 11
                Electronic Location Identifier: 102415
                Affiliations
                [a ]Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M DK-5230, Denmark
                [b ]Faculté des Sciences Site St Charles Aix Marseille Université, 3 place Victor Hugo, Marseille 13331, France
                Author notes
                [* ]Corresponding author. adelinar@ 123456bmb.sdu.dk
                Article
                Publisher Item ID: S2215-0161(23)00411-9 Publisher ID: 102415
                DOI: 10.1016/j.mex.2023.102415
                PMC ID: 10582779
                PubMed ID: 37860046
                SO-VID: 1da5844b-2fb1-48a9-b4b8-3accd5cb2904
                Copyright © © 2023 The Authors. Published by Elsevier B.V.
                License:

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 25 May 2023
                Date accepted : 4 October 2023
                Categories
                Subject: Biochemistry, Genetics and Molecular Biology

                Keywords: antigen retrieval and cryosectioning of multicellular 3d spheroids,spheroids,organoids,antigen retrieval,cryosectioning,immunostaining,fluorescent microscopy
                Data availability:
                Keywords: antigen retrieval and cryosectioning of multicellular 3d spheroids, spheroids, organoids, antigen retrieval, cryosectioning, immunostaining, fluorescent microscopy

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