Two‐Photon Imaging of the Immune System

Ivan L. Dzhagalov1, Heather J. Melichar1, Jenny O. Ross1, Paul Herzmark1, Ellen A. Robey1

1 Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.26
DOI:  10.1002/0471142956.cy1226s60
Online Posting Date:  April, 2012
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Abstract

Two‐photon microscopy is a powerful method for visualizing biological processes as they occur in their native environment in real time. The immune system uniquely benefits from this technology as most of its constituent cells are highly motile and interact extensively with each other and with the environment. Two‐photon microscopy has provided many novel insights into the dynamics of the development and function of the immune system that could not have been deduced by other methods and has become an indispensible tool in the arsenal of immunologists. In this unit, we provide several protocols for preparation of various organs for imaging by two‐photon microscopy that are intended to introduce the new user to some basic aspects of this method. Curr. Protoc. Cytom. 60:12.26.1‐12.26.20. © 2012 by John Wiley & Sons, Inc.

Keywords: two‐photon imaging; immune system; thymus; lymph node; gut; thymic slices

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparing the Thymus of a Mouse for Two‐Photon Imaging
  • Basic Protocol 2: Preparing the Mesenteric Lymph Nodes (MLNs) of a Mouse for Two‐Photon Imaging
  • Basic Protocol 3: Preparing Segments from the Intestine of a Mouse for Two‐Photon Imaging
  • Alternate Protocol 1: Agarose Embedding of a Small Tissue Sample or Organotypic Cultures
  • Alternate Protocol 2: Preparing Thymic Slices for Two‐Photon Imaging
  • Alternate Protocol 3: Overlaying Thymic Slices with Fluorescently Labeled Cells
  • Support Protocol: Setting Up Two‐Photon Imaging Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparing the Thymus of a Mouse for Two‐Photon Imaging

 Materials
  • Mouse for imaging (see Background Information)
  • 70% (v/v) ethanol spray (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010)
  • Tissue glue (Vetbond, 3M, cat. no. 149SB)
  • Styrofoam board
  • Tissue pins
  • Small scissors (Roboz, cat. no. RS‐5912)
  • Micro‐dissection scissors (Roboz, cat. no. RS‐5602)
  • Sharp forceps (Roboz, cat. no. RS‐5047)
  • 6‐cm tissue culture dishes (Corning, cat. no. 3516)
  • Paper towels
  • Kimwipes
  • Cover glasses (18‐mm circle; Fisherbrand, cat. no. 12‐545‐100)
  • Additional reagents and equipment for euthanizing the animal (Donovan and Brown, 2006)

Basic Protocol 2: Preparing the Mesenteric Lymph Nodes (MLNs) of a Mouse for Two‐Photon Imaging

 Materials
  • Mouse for imaging (see Background Information)
  • 70% (v/v) ethanol spray (see recipe)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010)
  • Tissue glue (Vetbond, 3M, cat. no. 149SB)
  • Styrofoam board
  • Tissue pins
  • Small scissors (Roboz, cat. no. RS‐5912)
  • Sharp forceps (Roboz, cat. no. RS‐5047)
  • Kimwipes or paper towels
  • Cover glasses (18‐mm circle; Fisherbrand, cat. no. 12‐545‐100)
  • 6‐cm tissue culture dishes (Corning, cat. no. 3516)
  • Additional reagents and equipment for euthanizing the animal (Donovan and Brown, 2006)

Basic Protocol 3: Preparing Segments from the Intestine of a Mouse for Two‐Photon Imaging

 Materials
  • Mouse for imaging (see Background Information)
  • 70% (v/v) ethanol spray (see recipe)
  • Ice‐cold complete RPMI 1640 medium (see recipe)
  • Tissue glue (Vetbond, 3M, cat. no. 149SB)
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010)
  • Styrofoam board
  • Tissue pins
  • Small scissors (Roboz, cat. no. RS‐5912)
  • 6‐cm tissue culture dishes (Corning, cat. no. 3516)
  • Cover glasses (18‐mm circle; Fisherbrand, cat. no. 12‐545‐100)
  • Micro‐dissection scissors (Roboz, cat. no. RS‐5602)
  • Additional reagents and equipment for euthanizing the animal (Donovan and Brown, 2006)

Alternate Protocol 1: Agarose Embedding of a Small Tissue Sample or Organotypic Cultures

 Materials
  • 4% low‐melting temperature agarose in HBSS (see recipe)
  • Crushed ice
  • Mouse fetal thymus, RTOC, or other small tissue sample in PBS
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010)
  • Tissue glue (3M Vetbond tissue adhesive, cat. no. 1469SB)
  • Foil
  • 500‐ml beakers
  • 37°C water bath
  • Molds for freezing tissue (7 × 7 × 5–mm disposable tissue molds; Curtin Matheson Scientific, cat. no. 038‐216)
  • Wide‐bore 1000‐µl pipet tips
  • 200‐µl pipet
  • Razor blade
  • Cover glasses (18‐mm circle; Fisherbrand, cat. no. 12‐545‐100 18CIR.‐1)
  • Spatula (Fisher Scientific, cat. no. 21‐401‐5), bent
  • 6‐cm tissue culture dishes (Corning, cat. no. 3516)

Alternate Protocol 2: Preparing Thymic Slices for Two‐Photon Imaging

 Materials
  • 4% low‐melting temperature agarose in HBSS (see recipe)
  • Crushed ice
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010)
  • Tissue glue (3M Vetbond tissue adhesive, cat. no. 1469SB)
  • 500‐ml beakers
  • Molds for freezing tissue (Polysciences, cat. no. 18986‐1)
  • Vibratome (1000 Plus Sectioning System)
  • Sharp blade (feather blades; Leica Biosystems, cat. no. 39053234)
  • 6‐cm tissue culture dishes (Corning, cat. no. 3516)
  • Spatula (Fischer Scientific, cat. no. 21‐401‐5), bent
  • Cover glasses (18‐mm circle; Fisherbrand, cat. no. 12‐545‐100 18CIR.‐1)
  • Additional reagents and equipment for harvesting the thymus (see Basic Protocol 1)

Alternate Protocol 3: Overlaying Thymic Slices with Fluorescently Labeled Cells

 Materials
  • Complete RPMI medium (see recipe)
  • Purified, fluorescently labeled cells of interest (e.g., CD4+ SP thymocytes) in medium
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010)
  • Tissue glue (3M Vetbond tissue adhesive, cat. no. 1469SB)
  • 6‐well plates (Corning)
  • 0.4‐µm pore‐size organotypic cell culture inserts (BD Biosciences, cat. no. 353090)
  • 200‐ and 20‐µl pipet tips
  • 200‐ and 20‐µl pipets
  • 37°C incubator
  • Cover glasses (18‐mm circle; Fisherbrand, cat. no. 12‐545‐100 18CIR.‐1)
  • Spatula (Fischer Scientific, cat. no. 21‐401‐5), bent
  • 6‐cm tissue culture dishes (Corning, cat. no. 3516)
  • Additional reagents and equipment for preparing the thymic slices (Alternate Protocol 2)

Support Protocol: Setting Up Two‐Photon Imaging Conditions

 Materials
  • Phenol red‐free DMEM (Mediatech, cat. no. 17‐205‐CV)
  • Vacuum grease (high‐vacuum grease; Dow Corning)
  • Oxygen (BioBlend 95% O2, 5% CO2 from PRAXAIR)
  • Cover glasses containing the samples (see Basic Protocol 1, 2, or 3 or Alternate Protocols 1, 2 or 3)
  • Imaging chamber
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Figures

  •  FigureFigure 12.26.1 Visualization of gluing intact tissue to the cover glass in preparation for two‐photon microscopy. (A) Add a drop of glue to the cover glass. (B) Limit the amount of glue on the cover glass to a small drop, as too much glue will negatively affect imaging. (C) Use forceps to grasp connective tissue, and carefully transfer the intact organ onto cover glass.
    View Image
  •  FigureFigure 12.26.2 Agarose embedding of small tissue pieces or organotypic culture for two‐photon imaging. (A) Transfer small tissue piece using a wide‐bore 1000‐µl tip in a small amount of PBS to the center of a tissue mold. (B) Carefully pour agarose solution over the tissue at the corner of the mold, making certain to avoid pouring agarose directly onto the tissue. (C) Allow the agarose to solidify on ice for ∼5 min. (D, E) Use a sharp blade to trim excess agarose around the edges of the mold. (F) After inverting the mold and pressing the agarose‐embedded tissue out, trim remaining excess agarose around and below the tissue. (G) Use a bent spatula to transfer agarose‐embedded tissue onto a cover glass with a small drop of glue (see also Fig. 12.26.1A,B). (H) Example of agarose‐embedded tissue on cover glass ready for two‐photon microscopy.
    View Image
  •  FigureFigure 12.26.3 Two‐photon microscopy of the thymus of a CD11c‐YFP+ mouse injected with Tomato lectin‐Texas Red (red) to label blood vessels, presented as a montage of maximal projections of adjacent three‐dimensional data sets spanning the cut thymus from the dorsal to the ventral side. The YFP+ dendritic cells (yellow) are concentrated in the medulla that is surrounded by cortex.
    View Image
  •  FigureFigure 12.26.4 Preparing thymic slices for direct two‐photon imaging or for the introduction of exogenous cells. (A) Place the thymus or similar tissue in a tissue mold with melted agarose solution and place on ice water. (B) After inverting the mold and pressing the agarose‐embedded tissue out, use a sharp blade to trim excess agarose. (C) Examples of horizontally (left) or vertically (right) agarose‐embedded tissue post‐trimming of extra agarose. Note that there is slightly more agarose on the bottom that is necessary for putting the whole tissue in the cutting range of the vibratome. (D) Agarose‐embedded tissue glued to vibratome stage. (E) As the tissue slices are cut with the vibratome, use a bent spatula to transfer them out of the vibratome stage. (F) To culture and/or add cells to the tissue slice, use a bent spatula to transfer to a cell culture insert in a 6‐well tissue culture dish, and carefully slide the tissue slice off of the spatula using a pipet tip. (G) Example of cut thymic slices on cell culture inserts where excess medium has been removed. Top, horizontally embedded and sliced thymus. Bottom, vertically embedded and sliced thymus. (H) For imaging, use a bent spatula to the transfer the tissue slice onto a cover glass that contains a small amount of glue (see also Fig. 12.26.1A,B).
    View Image

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