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Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation
BACKGROUND: Tissue fixation is crucial for preserving the morphology of biological structures and cytological details to prevent postmortem degradation and autolysis. Improper fixation conditions could lead to artifacts and thus incorrect conclusions in immunofluorescence or histology experiments. T...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320506/ https://www.ncbi.nlm.nih.gov/pubmed/25645690 http://dx.doi.org/10.1186/s12861-015-0056-y |
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author | Nie, Jing Mahato, Simpla Zelhof, Andrew C |
author_facet | Nie, Jing Mahato, Simpla Zelhof, Andrew C |
author_sort | Nie, Jing |
collection | PubMed |
description | BACKGROUND: Tissue fixation is crucial for preserving the morphology of biological structures and cytological details to prevent postmortem degradation and autolysis. Improper fixation conditions could lead to artifacts and thus incorrect conclusions in immunofluorescence or histology experiments. To resolve reported structural anomalies with respect to Drosophila photoreceptor cell organization we developed and utilized a combination of live imaging and fixed samples to investigate the exact biogenesis and to identify the underlying source for the reported discrepancies in structure. RESULTS: We found that piperazine-N,N’-bis(ethanesulfonic acid) (PIPES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), two zwitterionic buffers commonly used in tissue fixation, can cause severe lumen and cell morphological defects in Drosophila pupal and adult retina; the inter-rhabdomeral lumen becomes dilated and the photoreceptor cells are significantly reduced in size. Correspondingly, the localization pattern of Eyes shut (EYS), a luminal protein, is severely altered. In contrast, tissues fixed in the phosphate buffered saline (PBS) buffer results in lumen and cell morphologies that are consistent with live imaging. CONCLUSIONS: We suggest that PIPES and HEPES buffers should be utilized with caution for fixation when examining the interplay between cells and their extracellular environment, especially in Drosophila pupal and adult retina research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12861-015-0056-y) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4320506 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-43205062015-02-08 Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation Nie, Jing Mahato, Simpla Zelhof, Andrew C BMC Dev Biol Methodology Article BACKGROUND: Tissue fixation is crucial for preserving the morphology of biological structures and cytological details to prevent postmortem degradation and autolysis. Improper fixation conditions could lead to artifacts and thus incorrect conclusions in immunofluorescence or histology experiments. To resolve reported structural anomalies with respect to Drosophila photoreceptor cell organization we developed and utilized a combination of live imaging and fixed samples to investigate the exact biogenesis and to identify the underlying source for the reported discrepancies in structure. RESULTS: We found that piperazine-N,N’-bis(ethanesulfonic acid) (PIPES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), two zwitterionic buffers commonly used in tissue fixation, can cause severe lumen and cell morphological defects in Drosophila pupal and adult retina; the inter-rhabdomeral lumen becomes dilated and the photoreceptor cells are significantly reduced in size. Correspondingly, the localization pattern of Eyes shut (EYS), a luminal protein, is severely altered. In contrast, tissues fixed in the phosphate buffered saline (PBS) buffer results in lumen and cell morphologies that are consistent with live imaging. CONCLUSIONS: We suggest that PIPES and HEPES buffers should be utilized with caution for fixation when examining the interplay between cells and their extracellular environment, especially in Drosophila pupal and adult retina research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12861-015-0056-y) contains supplementary material, which is available to authorized users. BioMed Central 2015-02-03 /pmc/articles/PMC4320506/ /pubmed/25645690 http://dx.doi.org/10.1186/s12861-015-0056-y Text en © Nie et al.; licensee BioMed Central. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Article Nie, Jing Mahato, Simpla Zelhof, Andrew C Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation |
title | Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation |
title_full | Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation |
title_fullStr | Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation |
title_full_unstemmed | Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation |
title_short | Imaging the Drosophila retina: zwitterionic buffers PIPES and HEPES induce morphological artifacts in tissue fixation |
title_sort | imaging the drosophila retina: zwitterionic buffers pipes and hepes induce morphological artifacts in tissue fixation |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320506/ https://www.ncbi.nlm.nih.gov/pubmed/25645690 http://dx.doi.org/10.1186/s12861-015-0056-y |
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