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Histochemistry and Cell Biology

, Volume 144, Issue 6, pp 613–621 | Cite as

Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies

  • Raymond A. IsidroEmail author
  • Angel A. Isidro
  • Myrella L. Cruz
  • Siomara Hernandez
  • Caroline B. Appleyard
Original Paper

Abstract

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.

Keywords

Biotinylated IHC Macrophage Rat Ki-67 Double stain 

Notes

Acknowledgments

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).

Author contributions

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.

Ethical standard

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.

Supplementary material

418_2015_1364_MOESM1_ESM.mp4 (1.7 mb)
Online Resource 1 Double immunofluorescent staining with two mouse IgG1 antibodies (video) (MP4 1702 kb)
418_2015_1364_MOESM2_ESM.mp4 (2.1 mb)
Online Resource 2 Staining protocol that results in artifactual colocalization caused by interactions between tissue-bound anti-mouse secondary antibody and the biotinylated mouse antibody (video) (MP4 2102 kb)
418_2015_1364_MOESM3_ESM.pptx (1.6 mb)
Online Resource 3 Double immunofluorescent staining with antibodies from the same species in formalin-fixed paraffin-embedded colonic tissue from rats. Staining for the green (unconjugated primary antibody), red (biotinylated primary antibody), and blue channels is shown in the first, second, and third columns, respectively. Overlays of the three channels are shown in the fourth column. A–D, staining for CD68 (green), phosphorylated tyrosine (p-tyrosine, red), and DAPI (blue) in the inflamed colonic submucosa of a colitic rat shows several single- and double-positive cells for both CD68 and p-tyrosine. E–H, staining of matrix metalloprotease 9 (MMP9, green), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, red), and DAPI (blue) in the inflamed colonic submucosa of a colitic rat shows several MMP9 and GAPDH double-positive cells and many GAPDH single-positive cells. I–L, staining of vitamin D receptor (VDR, green), GAPDH (red), and DAPI (blue) in the colonic mucosa of a normal rat shows several VDR and GAPDH double-positive cells and GAPDH single-positive cells. Scale bars = 100 µm (PPTX 1651 kb)
418_2015_1364_MOESM4_ESM.pptx (2.1 mb)
Online Resource 4 Double immunofluorescent staining with antibodies from the same species in formalin-fixed paraffin-embedded colonic tissue from humans. Staining for the green (unconjugated primary antibody), red (biotinylated primary antibody), and blue channels is shown in the first, second, and third columns, respectively. Overlays of the three channels are shown in the fourth column. A–D, staining for CD68 (green), CD45RA (red), and DAPI (blue) in a mucosal lymphoid follicle of a normal human colon biopsy shows few CD68-positive cells (macrophages) in the center of the follicle surrounded by several CD45RA cells (lymphocytes). E–H, staining of vitamin D receptor (VDR, green), cleaved poly (ADP-ribose) polymerase (cPARP, red), and DAPI (blue) in the inflamed mucosa of a colonic biopsy from a patient with Crohn’s disease shows prevalent VDR staining in the lamina propria cells and crypt epithelium with scattered cPARP-positive cells. I–L, staining of VDR (green), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, red), and DAPI (blue) in the mucosa of a normal human colon biopsy shows several VDR and GAPDH double-positive cells. Scale bars = 100 µm (PPTX 2112 kb)
418_2015_1364_MOESM5_ESM.docx (109 kb)
Online Resource 5 Antibodies used in Online Resource 3 and Online Resource 4 (table) (DOCX 109 kb)
418_2015_1364_MOESM6_ESM.docx (119 kb)
Online Resource 6 Biotinylated antibodies that did not stain in formalin-fixed paraffin-embedded tissue by single immunofluorescence with fluorophore-conjugated streptavidin (table) (DOCX 118 kb)
418_2015_1364_MOESM7_ESM.pptx (445 kb)
Online Resource 7 Hematoxylin and eosin histochemical staining of areas of rat colon depicted in Figs. 2, 3, and 5. A, inflammatory infiltrate within colonic mucosa damaged by colitis induction as shown in Fig. 2. Scale bar = 100 µm. B, normal colonic mucosa and submucosa as shown in Fig. 3. Scale bar = 100 µm. C, crypts and lamina propria in normal colonic mucosa as shown in Fig. 5. Scale bar = 50 µm (PPTX 444 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Raymond A. Isidro
    • 1
    Email author
  • Angel A. Isidro
    • 1
  • Myrella L. Cruz
    • 1
  • Siomara Hernandez
    • 1
  • Caroline B. Appleyard
    • 1
  1. 1.Ponce Health Sciences University-Medical School and Ponce Research InstitutePonceUSA

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