The Membrane Marker mCLING Reveals the Molecular Composition of Trafficking Organelles

Natalia H. Revelo1, Silvio O. Rizzoli2

1 Department of Tumor Immunology, Institute for Molecular Life Sciences, Radboud University Medical Center, 2 Department of Neuro‐ and Sensory Physiology, University Medical Center Göttingen
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.25
DOI:  10.1002/0471142301.ns0225s74
Online Posting Date:  January, 2016
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Abstract

mCLING is a fixable endocytosis marker that can be combined with immunolabeling techniques to study the molecular composition of trafficking organelles. mCLING can be used both in cultured cells and in tissue if critical sample preparation steps, such as fixation, are correctly performed. This unit describes protocols for the application of mCLING and for the subsequent sample processing. We include immunostaining protocols and embedding procedures for confocal and high‐resolution microscopy. © 2016 by John Wiley & Sons, Inc.

Keywords: mCLING; membrane trafficking; endocytosis; high‐resolution microscopy; immunolabeling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: The Molecular Identity of Endocytosed Organelles in Cultured Cells
  • Alternate Protocol 1: Proteins Localized on the Plasma Membrane of Cells
  • Support Protocol 1: Antigen Retrieval After Fixation with 4% PFA + 0.2% Glutaraldehyde
  • Support Protocol 2: Preparation of Mowiol Embedding Medium
  • Support Protocol 3: Preparation of Melamine Solution and Embedding Procedure
  • Basic Protocol 2: Using mCLING in Tissue Preparations
  • Support Protocol 4: Embedding in Thiodiethanol (TDE)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: The Molecular Identity of Endocytosed Organelles in Cultured Cells

  Materials
  • Cells cultured on coverslips (these can be primary cultured neurons or any stable cell line of interest; e.g., COS‐7, HEK293, PC12 cells)
  • Physiological buffered saline (e.g., Tyrode's buffer for cultured neurons and Ringer's buffer for cultured cell lines like COS‐7; see reciperecipes)
  • mCLING lyophilized (Synaptic Systems, cat. no. 710 006): Resuspend in 100 μl of H 2O (final concentration 50 μM; conjugations available with Atto 647N, Atto 488 and Dy‐654)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Fixative solution: 4% PFA + 0.2% glutaraldehyde in phosphate‐buffered saline (PBS; see recipe)
  • Ice
  • Quenching solution: 100 mM NH 4Cl + 100 mM Glycine in PBS
  • Permeabilization and blocking solution: 0.1% Triton X‐100 + 2.5% bovine serum albumin (BSA) in PBS
  • Primary antibody of choice
  • Secondary antibody
  • High‐salt phosphate‐buffered saline (PBS; see recipe)
  • Mowiol (for direct embedding of coverslips, see protocol 4)
  • Melamine solution (for resin embedding and further sectioning, see protocol 5)
  • 37°C incubator
  • Coverslips
  • Ultramicrotome (required for melamine blocks slicing), optional
NOTE: The volumes provided in this protocol are suggested for cell cultures on coverslips with 18‐mm of diameter, incubated in a 12‐well plate.

Alternate Protocol 1: Proteins Localized on the Plasma Membrane of Cells

  Additional Materials (also see Basic Protocol)
  • 100‐ml beakers
  • 12‐well plates (or any other convenient format)

Support Protocol 1: Antigen Retrieval After Fixation with 4% PFA + 0.2% Glutaraldehyde

  Additional Materials (also see protocol 1)
  • Test cells
  • Basic antigen retrieval solution (R&D Systems, cat. no. CTS013)
  • Fixative solution:
    • 4% paraformaldehyde (PFA) in phoshate‐buffered saline (PBS)
    • 4% PFA + 0.2% glutaraldehyde
  • Phosphate‐buffered saline (PBS; see recipe)
  • Water bath
  • 12‐well plate (or any other format of your convenience)
NOTE: The volumes indicated in this protocol are suggested for coverslips with 18‐mm diameter incubated in a 12‐well plate.

Support Protocol 2: Preparation of Mowiol Embedding Medium

  Materials
  • Glycerol
  • Mowiol 4‐88 reagent (Merck Millipore)
  • Distilled water
  • 1 M Tris buffer
  • Stirring hotplate
  • Stir bar
  • Parafilm
  • 1.5‐ml tubes
  • Oven

Support Protocol 3: Preparation of Melamine Solution and Embedding Procedure

  Materials
  • p‐Toluensulfonic acid monohydrate (Sigma‐Aldrich, cat. no. 402885); catalyzer for the polymerization of melamine
  • Distilled water
  • 2,4,6‐Tris[bis(methoxymethyl)amino]‐1,3,5‐triazine (melamine, TCI Europe, cat. no. T2059)
  • Silica gel type III (Sigma‐Aldrich, cat. no. S7625)
  • Epon resin (Epo Fix kit, Stuers, cat. no. 40200029)
  • Mowiol
  • 15‐ml conical tubes
  • Vortex mixer
  • Benchtop horizontal shaker
  • BEEM capsules (Beem, cat. no. 1001, Size 00)
  • Plastic petri dish lids (60mm diameter)
  • Plastic boxes (IBI Scientific, cat. no. 10066‐576)
  • Oven
  • Metal blades
  • Ultramicrotome
  • Heating plate

Basic Protocol 2: Using mCLING in Tissue Preparations

  Materials
  • Muscle or synaptic preparation, freshly dissected
  • Physiological‐buffered saline (for electrical stimulation): recommended solutions are Hank's balanced salt solution for tissues like the organ of Corti, mouse buffer for mouse NMJs, or Jan & Jan's buffer for Drosophila NMJs [see reciperecipes; for stimulation via membrane depolarization, solutions can be prepared with a high concentration of K+ (40 to 70 mM, reduce Na+ concentration accordingly)]
  • Physiological‐buffered saline without Ca2+ (same recipe but do not add Ca2+)
  • mCLING lyophilized (Synaptic Systems, cat. no. 710 006): resuspend in 100 μl of H 2O (final concentration 50 μM)
  • Fixative solution: 4% paraformaldehyde (PFA) + 0.2% glutaraldehyde in phosphate‐buffed saline (PBS; see recipe)
  • Ice
  • Phosphate‐buffered saline (PBS; see recipe)
  • Quenching solution: 100 mM NH 4Cl + 100 mM Glycine in PBS
  • Permeabilization and blocking solution: 0.5% Triton X‐100 + 2.5% bovine serum albumin (BSA) in PBS
  • Primary antibody of choice
  • Secondary antibody of choice
  • High‐salt phosphate‐buffered saline (PBS; see recipe)
  • Melamine solution (for tissue embedding and further sectioning, see protocol 5)
  • Small petri dish lids (Falcon 35‐mm diameter; Corning, cat. no. 353001) lined to half of the volume with Sylgard 184 Silicone elastomer (Dow Corning); optional, but strongly recommended for Drosophila larvae and mammalian muscle preparations
  • Dissection pins (stainless steel, no. 10)
  • Dissection blade or the sharp end of a small needle
  • Glass coverslips 18‐mm diameter no. 1 (Menzel‐Glässer, cat. no. CB00180RA1)
  • Forceps
  • 60‐mm diameter plastic petri dishes
  • Micropipets
  • Whatman filter paper cut into small triangles (2 cm × 2 cm × 1 cm)
  • PLL‐coated coverslips (optional, to favor adhesion of hard tissues)

Support Protocol 4: Embedding in Thiodiethanol (TDE)

  Materials
  • 2,2’‐Thiodiethanol (TDE; Sigma‐Aldrich, cat. no. 166782)
  • Distilled water
  • Picodent twinsil speed dental resin (components A and B, Dental‐Produktions‐ und Vertriebs‐GmbH)
  • Horizontal shaker
  • Micropipets
  • 18‐ and 30‐mm diameter glass coverslips
  • Dissection microscope
  • Fine forceps and fine needles
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Figures

Videos

Literature Cited

Literature Cited
  Betz, W.J., Mao, F., and Bewick, G.S. 1992. Activity‐dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J. Neurosci. 12:363‐375.
  Ceccarelli, B., Hurlbut, W.P., and Mauro, A. 1973. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. J. Cell Biol. 57:499‐524. doi: 10.1083/jcb.57.2.499.
  Coon, A.H., Creech, H.J., Jones, N., and Berliner, E. 1942. The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J. Immunol. 45:159‐170.
  Coons, A.H., Creech, H.J., and Jones, R.N. 1941. Immunological properties of an antibody containing a fluorescent group. Proc. Soc. Exp. Biol. Med. 47:200‐202. doi: 10.3181/00379727‐47‐13084P.
  Denker, A., Bethani, I., Kröhnert, K., Körber, C., Horstmann, H., Wilhelm, B.G., Barysch, S. V, Kuner, T., Neher, E., and Rizzoli, S.O. 2011. A small pool of vesicles maintains synaptic activity in vivo. Proc. Natl. Acad. Sci. U.S.A. 108:17177‐17182. doi: 10.1073/pnas.1112688108.
  Faulk, W. and Taylor, G. 1971. An immunocolloidal method for the electron microscope. Immunochemistry 8:1081‐1083. doi: 10.1016/0019‐2791(71)90496‐4.
  Fawcett, D.W. 1965. Surface specializations of absorbing cells. J. Histochem. Cytochem. 13:75‐91. doi: 10.1177/13.2.75.
  Hell, S.W. 2007. Far‐field optical nanoscopy. Science 316:1153‐1158. doi: 10.1126/science.1137395.
  Henkel, A.W., Lübke, J., and Betz, W.J. 1996. FM1‐43 dye ultrastructural localization in and release from frog motor nerve terminals. Proc. Natl. Acad. Sci. U.S.A. 93:1918‐1923. doi: 10.1073/pnas.93.5.1918.
  Heuser, J.E. and Reese, T.S. 1973. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol. 57:315‐344. doi: 10.1083/jcb.57.2.315.
  Heuser, J.E., Reese, T.S., Dennis, M.J., Jan, Y., Jan, L., and Evans, L. 1979. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J. Cell Biol. 81:275‐300. doi: 10.1083/jcb.81.2.275.
  Honig, M.G. and Hume, R.I. 1986. Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long‐term cultures. J. Cell Biol. 103:171‐187. doi: 10.1083/jcb.103.1.171.
  Jan, L.Y. and Jan, Y.N. 1976. Properties of the larval neuromuscular junction in Drosophila melanogaster. J. Physiol. 262:189‐214. doi: 10.1113/jphysiol.1976.sp011592.
  Klausner, R.D. and Wolf, D.E. 1980. Selectivity of fluorescent lipid analogues for lipid domains. Biochemistry 19:6199‐6203. doi: 10.1021/bi00567a039.
  Lichtman, J.W. and Wilkinson, R.S. 1987. Properties of motor units in the transversus abdominis muscle of the garter snake. J. Physiol. 393:355‐374. doi: 10.1113/jphysiol.1987.sp016827.
  Malatesta, M., Zancanaro, C., Costanzo, M., Cisterna, B., and Pellicciari, C. 2013. Simultaneous ultrastructural analysis of fluorochrome‐photoconverted diaminobenzidine and gold immunolabelling in cultured cells. Eur. J. Histochem. 57:e26:168‐171. doi: 10.4081/ejh.2013.e26.
  Marrack, J. 1934. Nature of antibodies. Nature 133:292‐293. doi: 10.1038/133292b0.
  Opazo, F., Levy, M., Byrom, M., Schäfer, C., Geisler, C., Groemer, T.W., Ellington, A.D., and Rizzoli, S.O. 2012. Aptamers as potential tools for super‐resolution microscopy. Nat. Methods 9:938‐939. doi: 10.1038/nmeth.2179.
  Perkel, J.M. 2014. The antibody challenge. Biotechniques 56:111‐114.
  Revelo, N.H. 2014. A novel membrane‐binding probe for the morphological and molecular characterization of synaptic vesicle recycling pathways. Ph.D. dissertation. Georg‐August‐Universität Göttingen, Germany. http://hdl.handle.net/11858/00‐1735‐0000‐0022‐5FDB‐C.
  Revelo, N.H. and Rizzoli, S.O. 2015. Application of STED microscopy to cell biology questions. Methods Mol. Biol. 1251:213‐230. doi: 10.1007/978‐1‐4939‐2080‐8_12.
  Revelo, N.H., Kamin, D., Truckenbrodt, S., Wong, A.B., Reuter‐Jessen, K., Reisinger, E., Moser, T., and Rizzoli, S.O. 2014. A new probe for super‐resolution imaging of membranes elucidates trafficking pathways. J. Cell Biol. 205:591‐606. doi: 10.1083/jcb.201402066.
  Ries, J., Kaplan, C., Platonova, E., Eghlidi, H., and Ewers, H. 2012. A simple, versatile method for GFP‐based super‐resolution microscopy via nanobodies. Nat. Methods: 9:582‐584. doi: 10.1038/nmeth.1991.
  Sandell, J.H. and Masland, R.H. 1988. Photoconversion of some fluorescent markers to a diaminobenzidine product. J. Histochem. Cytochem. 36:555‐559. doi: 10.1177/36.5.3356898.
  Singer, S.J. 1959. Preparation of an electron‐dense antibody conjugate. Nature 183:1523‐1524. doi: 10.1038/1831523a0.
  Spiegel, S., Kassis, S., Wilchek, M., and Fishman, P.H. 1984. Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes. J. Cell Biol. 99:1575‐1581. doi: 10.1083/jcb.99.5.1575.
  Staudt, T., Lang, M.C., Medda, R., Engelhardt, J., and Hell, S.W. 2007. 2,2’‐thiodiethanol: A new water soluble mounting medium for high resolution optical microscopy. Microsc. Res. Tech. 70:1‐9. doi: 10.1002/jemt.20396.
  Struck, D.K. and Pagano, R.E. 1980. Insertion of fluorescent phospholipids into the plasma membrane of a mammalian cell. J. Biol. Chem. 255:5404‐5410.
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