TY - JOUR
T1 - Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling
AU - Forsthoefel, David J.
AU - Waters, Forrest A.
AU - Newmark, Phillip A.
N1 - Funding Information:
We thank Amanda Adams, Ryan King, Marla Tharp, Amir Saberi, Bo Wang, and John Brubacher (Canadian Mennonite University) for critical readings of the manuscript, and all members of the Newmark Lab for thoughtful discussions. We are grateful to Ricardo Zayas and Kelly Ross (San Diego State University) for sharing results from an independently conducted antibody screen in advance of publication. We gratefully acknowledge Liping Wang, Rachel Breitenfeld, and Xiaoxia Wang of the University of Illinois Immunological Resources Facility for exceptional technical advice and contributions for the duration of the project. We are also grateful to Karla Daniels of the Developmental Studies Hybridoma Bank at the University of Iowa for her advice on supernatant storage and for kindly supplying test aliquots. This work was supported by a Ruth L. Kirschstein National Research Service Award from the National Institutes of Health (F32-DK077469) to DJF and by NIH grant R01-HD043403 to PAN. PAN is an investigator of the Howard Hughes Medical Institute.
Publisher Copyright:
© 2014 Forsthoefel et al.; licensee BioMed Central.
PY - 2014
Y1 - 2014
N2 - Background: Efforts to elucidate the cellular and molecular mechanisms of regeneration have required the application of methods to detect specific cell types and tissues in a growing cohort of experimental animal models. For example, in the planarian Schmidtea mediterranea, substantial improvements to nucleic acid hybridization and electron microscopy protocols have facilitated the visualization of regenerative events at the cellular level. By contrast, immunological resources have been slower to emerge. Specifically, the repertoire of antibodies recognizing planarian antigens remains limited, and a more systematic approach is needed to evaluate the effects of processing steps required during sample preparation for immunolabeling. Results: To address these issues and to facilitate studies of planarian digestive system regeneration, we conducted a monoclonal antibody (mAb) screen using phagocytic intestinal cells purified from the digestive tracts of living planarians as immunogens. This approach yielded ten antibodies that recognized intestinal epitopes, as well as markers for the central nervous system, musculature, secretory cells, and epidermis. In order to improve signal intensity and reduce non-specific background for a subset of mAbs, we evaluated the effects of fixation and other steps during sample processing. We found that fixative choice, treatments to remove mucus and bleach pigment, as well as methods for tissue permeabilization and antigen retrieval profoundly influenced labeling by individual antibodies. These experiments led to the development of a step-by-step workflow for determining optimal specimen preparation for labeling whole planarians as well as unbleached histological sections. Conclusions: We generated a collection of monoclonal antibodies recognizing the planarian intestine and other tissues; these antibodies will facilitate studies of planarian tissue morphogenesis. We also developed a protocol for optimizing specimen processing that will accelerate future efforts to generate planarian-specific antibodies, and to extend functional genetic studies of regeneration to post-transcriptional aspects of gene expression, such as protein localization or modification. Our efforts demonstrate the importance of systematically testing multiple approaches to species-specific idiosyncracies, such as mucus removal and pigment bleaching, and may serve as a template for the development of immunological resources in other emerging model organisms.
AB - Background: Efforts to elucidate the cellular and molecular mechanisms of regeneration have required the application of methods to detect specific cell types and tissues in a growing cohort of experimental animal models. For example, in the planarian Schmidtea mediterranea, substantial improvements to nucleic acid hybridization and electron microscopy protocols have facilitated the visualization of regenerative events at the cellular level. By contrast, immunological resources have been slower to emerge. Specifically, the repertoire of antibodies recognizing planarian antigens remains limited, and a more systematic approach is needed to evaluate the effects of processing steps required during sample preparation for immunolabeling. Results: To address these issues and to facilitate studies of planarian digestive system regeneration, we conducted a monoclonal antibody (mAb) screen using phagocytic intestinal cells purified from the digestive tracts of living planarians as immunogens. This approach yielded ten antibodies that recognized intestinal epitopes, as well as markers for the central nervous system, musculature, secretory cells, and epidermis. In order to improve signal intensity and reduce non-specific background for a subset of mAbs, we evaluated the effects of fixation and other steps during sample processing. We found that fixative choice, treatments to remove mucus and bleach pigment, as well as methods for tissue permeabilization and antigen retrieval profoundly influenced labeling by individual antibodies. These experiments led to the development of a step-by-step workflow for determining optimal specimen preparation for labeling whole planarians as well as unbleached histological sections. Conclusions: We generated a collection of monoclonal antibodies recognizing the planarian intestine and other tissues; these antibodies will facilitate studies of planarian tissue morphogenesis. We also developed a protocol for optimizing specimen processing that will accelerate future efforts to generate planarian-specific antibodies, and to extend functional genetic studies of regeneration to post-transcriptional aspects of gene expression, such as protein localization or modification. Our efforts demonstrate the importance of systematically testing multiple approaches to species-specific idiosyncracies, such as mucus removal and pigment bleaching, and may serve as a template for the development of immunological resources in other emerging model organisms.
KW - Immunofluorescence
KW - Immunohistochemistry
KW - Intestine
KW - Monoclonal antibody screen
KW - Planarian
KW - Regeneration
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U2 - 10.1186/s12861-014-0045-6
DO - 10.1186/s12861-014-0045-6
M3 - Article
C2 - 25528559
AN - SCOPUS:84937574057
SN - 1471-213X
VL - 14
JO - BMC Developmental Biology
JF - BMC Developmental Biology
M1 - 45
ER -