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 1: 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, )

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, )

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, )

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 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 ( protocol 5)

Support Protocol 1: 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% O 2, 5% CO 2 from PRAXAIR)
  • Cover glasses containing the samples (see protocol 1, 2, or 3 or Alternate Protocols protocol 41, protocol 52 or protocol 63)
  • Imaging chamber
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Figures

Literature Cited

Literature Cited
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