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Protocol for the Isolation and Super-resolution dSTORM Imaging of RyR2 in Cardiac Myocytes

YH Yufeng Hou
CL Christopher Le
CS Christian Soeller
WL William E. Louch

Published: Aug 5, 2018 DOI: 10.21769/BioProtoc.2952 Views: 5408

Edited by: Joe Zhang Reviewed by: Anca Savulescu

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Abstract

Since its inception, super-resolution microscopy has played an increasingly important role in the discovery and characterization of nanoscale biological structure. dSTORM, which is one of the most commonly applied methods, relies on stochastic photoswitching of fluorophores to recreate a super-resolution image. The cardiac field has particularly benefitted from the application of this technique, as it has enabled sub-diffraction-limit visualization of calcium release units (CRUs) and the fundamental structures that trigger contraction. Acquisition of such images requires careful, reproducible sample preparation, and consistent imaging conditions maintained for the duration of the experiment. Here we present standardized methods for the production of dSTORM images of the Ca2+ release channel Ryanodine Receptor type-2 (RyR2) in cardiac myocytes. The presented protocols specifically focus on steps involved in primary cardiac myocyte isolation, sample preparation, and imaging with details provided for experimental solutions and microscope settings. This discussion is followed by an overview of various analysis techniques to discern RyR2 organization within clusters and CRUs.

Keywords: Super-resolution search dSTORM search Langendorff search Cell Isolation search Image processing search

Background

In recent years, super-resolution microscopy has seen a rapid rise in popularity. A variety of super-resolution techniques have been described which enable optical resolution well below the diffraction limit of light, in some cases approaching that obtainable by electron microscopy. Together, the advent of these techniques has led to an explosion of new research into nanoscale biological structure, domains, and protein interactions. One popular super-resolution technique is direct Stochastic Optical Microscopy (dSTORM), which pairs the benefits of relatively simple sample handling with an ~10x improvement in resolution in comparison with standard confocal microscopy (van de Linde et al., 2011). The trade-off, however, is an increased acquisition time as well as complexity of analysis which can seem daunting to those starting in the field. While recent commercial systems from the major imaging companies such as Zeiss, Nikon, and Olympus have made dSTORM more accessible to biologists, the technique still requires careful planning of experiments and accurate, reproducible protocols to ensure the production of high-quality images.

The Ryanodine Receptor type 2 (RyR2) protein is a homo-tetrameric Ca2+ release channel localized within the sarcoplasmic reticulum of cardiac myocytes, which is an important target for super-resolution structural studies (Jayasinghe et al., 2012; Soeller and Baddeley, 2013; Asghari et al., 2014; Hiess et al., 2015; Hou et al., 2015; Munro et al., 2016). Indeed, the RyR is well-suited to such studies, owing to its large size and its tendency to agglomerate into functionally important ‘clusters’. Most of these clusters have sizes that are just below the resolution of conventional microscopes. Because of these desirable features, the RyR can also serve as a useful example protein for introducing methods in sample preparation and imaging in a more general context. Here we present standardized methods to produce high-quality dSTORM images using the Carl Zeiss Elyra P1 dSTORM setup, with the RyR2 as a model target. The outlined protocols include methods for primary cardiac myocyte isolation, sample preparation, and imaging. Further discussion is provided regarding the analysis of RyR2 organization, including techniques for discernment of RyR clusters and, in turn, Ca2+ Release Units (CRUs) which are functional groupings of RyR clusters thought to underlie Ca2+ sparks (Inui et al., 1987).

Materials and Reagents

  1. Consumables
    1. 18 gauge disposable needle (BD, catalog number: 301900 )
    2. Weigh boat
    3. Falcon tubes 50 ml, 15 ml (Corning, catalog numbers: 430829 , 430791 )
    4. Pasture pipette (VWR, catalog number: VWRI612-1684 )
    5. 1.5 Coverslips on dish (MATTEK, catalog number: P35G-0.170-14-C )
      Coverslip preparation: Coverslips for the experiment also require prior preparation. To obtain very clean coverslips, we wash coverslips first with EtOH and allow them to dry. Apply 200 μl of prepared Poly-L-Lysine (0.01%) solution to the coverslip and incubate overnight at 4 °C or 2 h at 37 °C. The Poly-L Lysine coating allows cell adhesion when plated.

  2. Animals
    1. Mice (Breed: C57BL/6J)

  3. Reagents
    1. EtOH (VWR, catalog number: 20824.296 )
    2. NaCl (Sigma-Aldrich, catalog number: 71376 )
    3. KCl (Sigma-Aldrich, catalog number: P9541 )
    4. HEPES (Sigma-Aldrich, catalog number: H3375 )
    5. MgCl2·6H2O (Sigma-Aldrich, catalog number: M2393 )
    6. NaH2PO4 (Sigma-Aldrich, catalog number: S5011 )
    7. D-Glucose (Sigma-Aldrich, catalog number: 49159 )
    8. Isoflurane (Abbott, catalog number: B506 )
    9. Bovine serum albumin (BSA) (Stock dilution: 2 g/100 ml)
    10. DNase, Batch Number: 57B17285 (Worthington Biochemical, catalog number: LS002006 )
    11. Phosphate buffered saline (PBS) (Lonza, BioWhittakerTM, catalog number: BE17-512F )
    12. Collagenase 2, Activity 265 units, Batch number: 45A15450 (Worthington Biochemical, catalog number: LS004176 )
    13. Bovine Serum Albumin (powder) (Sigma-Aldrich, catalog number: A2153 )
    14. CaCl2 (Sigma-Aldrich, catalog number: 449709 )
    15. PFA (Electron Microscopy Sciences, catalog number: 19208 )
    16. Glycine (Sigma-Aldrich, catalog number: G7126 )
      Note: Prepare Glycine 100 mM in PBS. 
    17. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
      Note: Prepare 0.03% Triton X-100 (v/v) in PBS.
    18. Image iT FX signal Enhancer (Thermo Fisher Scientific, catalog number: I36933 )
    19. Poly-L-lysine solution 0.01% w/v (Sigma-Aldrich, catalog number: P4707 )
    20. Low Blocking Buffer (Thermo Fisher Scientific, catalog number: 00-4953-54 )
    21. NaOH (Sigma-Aldrich, catalog number: S5881 )
    22. Immersion oil 30 degrees (Carl Zeiss, catalog number: Immersol-518F , 30 °C)
    23. Cell isolation buffer (CIV) (see Recipes for composition)
    24. Collagenase solution (see Recipes for composition)
    25. 4% Paraformaldehyde (PFA) solution (see Recipes for composition)

  4. Antibodies
    1. Mouse Anti-RyR1 Primary antibody (Thermo Fisher Scientific, catalog number: MA3-916 )
    2. Donkey Anti-mouse Alexa 647 Secondary (Abcam, catalog number: ab181292 )

  5. Mounting media
    1. Vecta Shield (Vector Laboratories, catalog number: H-1000 )
    2. Glycerol solution 86-89% (Sigma-Aldrich, catalog number: 49781 )

Equipment

  1. Mechanical apparatus
    1. Peristaltic perfusion pump (Watson-Marlow Fluid Technology Group, model: 101U )
    2. Standard gauge infusion tubes x3 with added three-way valve
    3. Water bath (37 °C) with pump to water jacket
    4. Microdissection kit
      1. Scissors
      2. Fine tip forceps
      3. Curved scissors
      4. Curved tip forceps
    5. Heating plate
    6. Measuring cylinder 500 ml
    7. Schott Bottle 500 ml
    8. Pipettes–10 μl, 200 μl, 1,000 μl (Thermo Fisher Scientific, Finnpipette)
    9. Scales (Sartorius, models: BL310 and CP224S )
    10. pH meter (Radiometer Copenhagen, model: pHM 92 )

  2. Imaging
    1. Carl Zeiss LSM 710 inverted confocal microscope (Carl Zeiss, model: LSM 710 )
    2. Carl Zeiss ELYRA P1 dSTORM attachment for LSM 710 (Carl Zeiss, model: ELYRA P.1 ) Supplied with:
      1. PLAN Apocromat 63x NA 1.41 Oil Immersion objective
      2. PLAN Apocromat 100x NA 1.46 Oil Immersion objective
      3. ANDOR Ixon EMCCD
      4. Halogen lamp
      5. TIRF filter set (SIM, TIRF, TIRF-HP, TIRF-uHP)
      6. 200 mW 642 nm Laser and associated filters

Software

  1. Zen Black software (Carl Zeiss)
  2. ImageJ Software

Procedure

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Cell Biology > Cell isolation and culture > Cell isolation

Cell Biology > Cell imaging > Confocal microscopy

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