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Optimized protocol for immunostaining of experimental GFP-expressing and human hearts

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Abstract

Morphological and histochemical analysis of the heart is fundamental for the understanding of cardiac physiology and pathology. The accurate detection of different myocardial cell populations, as well as the high-resolution imaging of protein expression and distribution, within the diverse intracellular compartments, is essential for basic research on disease mechanisms and for the translatability of the results to human pathophysiology. While enormous progress has been made on the imaging hardware and methods and on biotechnological tools [e.g., use of green fluorescent protein (GFP), viral-mediated gene transduction] to investigate heart cell structure and function, most of the protocols to prepare heart tissue samples for analysis have remained almost identical for decades. We here provide a detailed description of a novel protocol of heart processing, tailored to the simultaneous detection of tissue morphology, immunofluorescence markers and native emission of fluorescent proteins (i.e., GFP). We compared a variety of procedures of fixation, antigen unmasking and tissue permeabilization, to identify the best combination for preservation of myocardial morphology and native GFP fluorescence, while simultaneously allowing detection of antibody staining toward sarcomeric, membrane, cytosolic and nuclear markers. Furthermore, with minimal variations, we implemented such protocol for the study of human heart samples, including those already fixed and stored with conventional procedures, in tissue archives or bio-banks. In conclusion, a procedure is here presented for the laboratory investigation of the heart, in both rodents and humans, which accrues from the same tissue section information that would normally require the time-consuming and tissue-wasting observation of multiple serial sections.

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Abbreviations

AAV9:

Adeno-associated virus serotype 9

α/βMyHC:

α/β-Myosin Heavy Chain

BSA:

Bovine serum albumin

CHMP2B:

Charged multivesicular body protein 2B

Cx43:

Connexin-43

EGFP:

Enhanced green fluorescent protein

FF-PE:

Formalin-fixed paraffin-embedded

FP:

Fluorescent protein

GFP:

Green fluorescent protein

HHT:

Heterotopic heart transplant

IF:

Immunofluorescence

PBS:

Phosphate-buffered saline

PFA:

Paraformaldehyde

SMA:

Smooth muscle actin

WT:

Wild type

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Acknowledgments

We thank Dr. Stefano Schiaffino for critical reading of the manuscript and support to the experiments; Drs Emanuele Cozzi, Arben Dedja and Luigi Dall’Olmo for heterotopic heart transplantation. We thank Dr Camillo Barbisan for assistance on medical legal issues inherent in the use of human samples. We are also grateful to Alessandra Dubrovich and Emilio Bigon for technical assistance. This work was supported by University of Padova (Progetto Giovani Studiosi 2010, contract: GRIC101133) to Tania Zaglia, Telethon-Italy (GGP11224) to Marco Mongillo.

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    Authors and Affiliations

    1. Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/b, 35133, Padua, Italy

      Tania Zaglia, Simonetta Ausoni & Marco Mongillo

    2. Venetian Institute of Molecular Medicine (VIMM), Via Orus 2, 35129, Padua, Italy

      Tania Zaglia, Anna Di Bona & Marco Mongillo

    3. Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Via A. Gabelli, 61, 35121, Padua, Italy

      Anna Di Bona & Cristina Basso

    4. Istituto Comprensivo di Carmignano di Brenta, Padua, Italy

      Tatiana Chioato

    5. CNR Institute of Neuroscience, Viale G. Colombo 3, 35121, Padua, Italy

      Marco Mongillo

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    1. Tania Zaglia
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    2. Anna Di Bona
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    4. Cristina Basso
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    5. Simonetta Ausoni
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    Corresponding author

    Correspondence to Tania Zaglia.

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    The authors declare no conflicting of interest.

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    Simonetta Ausoni and Marco Mongillo have contributed equally to this article.

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    Figure 1

    Effects of the combined ‘Microwave+Triton’ treatment on tissue autofluorescence. (a–b) Hearts from GFP+ (a) and WT (b) rats were fixed by PFA perfusion. Ten µm heart sections underwent ‘microwave+Triton’ unmasking procedure and were imaged at the fluorescence microscopy using the same acquisition parameters. The images evidence that our protocol significantly blunts tissue autofluorescence without interfering with the GFP signal. (TIFF 3480 kb)

    Figure 2

    Secondary antibody staining in GFP positive mouse and rat heart sections. (a–c) Ventricular sections from GFP positive mice (a) and rats (b–c) underwent ‘microwave+Triton’ unmasking procedure, and were treated with 1X PBS, supplemented with 1% BSA and 1% Triton-X100 without a primary antibody, followed by incubation with anti-rabbit (a-b) and anti-mouse (c) secondary antibodies. (TIFF 14639 kb)

    Figure 3

    Validation of the new immunofluorescence protocol for the analysis of GFP positive mouse heart. (a–d) Immunofluorescence and microscope analysis on serial ventricular sections which underwent: no treatment (NT); microwave (Mw); Triton (Tr) or ‘microwave+Triton’ (Mw/Tr) treatments. Heart sections were stained with antibodies to: α/β-Myosin Heavy Chain (α/β-MHC) (a); desmin (b); GATA-4 (c); connexin-43 (d, cx-43). The signal of the native EGFP fluorescence upon the combination of Mw/Tr treatments is shown in the right panels (green signal). The white arrow in (c) evidences a GATA-4 positive cardiomyocyte nucleus. (TIFF 17504 kb)

    Figure 4

    Tissue morphology in heart sections from experimental models processed with the new immunofluorescence protocol. (a–d) Haematoxyln-eosin staining was performed in ventricular sections from heterotopic heart transplants (a–b) and hearts infected with AAV9-GFP-U6-CHMP2Bsh (c–d), before (a and c) and after (b and d) ‘microwave+Triton’ treatment. (TIFF 16867 kb)

    Figure 5

    Secondary antibody staining in human heart sections. (a–b) FF-PE heart sections underwent ‘microwave+Triton’ treatment and incubation with Cy3 conjugated anti-mouse (a), 488-conjugated anti-rabbit (b) and Cy3-conjugated anti-rabbit secondary antibodies, without previous incubation with specific primary antibodies. Sections were analyzed at the fluorescence microscopy. (TIFF 12676 kb)

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    Zaglia, T., Di Bona, A., Chioato, T. et al. Optimized protocol for immunostaining of experimental GFP-expressing and human hearts. Histochem Cell Biol 146, 407–419 (2016). https://doi.org/10.1007/s00418-016-1456-1

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