Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies
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The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated.
KeywordsBiotinylated IHC Macrophage Rat Ki-67 Double stain
The authors would like to thank Alcira Benitez Barros for histotechnical assistance. We also thank Dr. Pedro Santiago for comments on the manuscript. RA Isidro was supported by a William Townsend Porter Predoctoral Fellowship from the American Physiological Society. This study was also funded in part by the National Institute of General Medical Sciences (R25GM082406 to RAI, SH) and the National Cancer Institute (U54CA163071 to RAI, CBA) of the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors acknowledge the support of the PHSU Molecular and Genomics Core Laboratory (RR003050/MD007579).
RAI, AAI, and CBA designed the study and analyzed the data. RAI, MLC, and SH performed the experiments and acquired the data. RAI drafted the manuscript. AAI, MLC, SH, and CBA revised manuscript for important intellectual content. RAI, AAI, MLC, SH, and CBA approved the final version of the manuscript for publication and agree to be accountable for all aspects of the work.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted. Informed consent was obtained from all individual participants included in this study.
Human and animal rights
All animal studies were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Ponce Health Sciences University and of the National Institutes of Health. All human studies were performed in accordance with the guidelines of the Internal Review Board at Ponce Health Sciences University and of the National Institutes of Health.
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