Laser Microdissection

Andra R. Frost1, Isam‐Eldin Eltoum1, Gene P. Siegal1, Michael R. Emmert‐Buck2, Michael A. Tangrea3

1 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, 2 Avoneaux Medical Institute, Oxford, Maryland, 3 Alvin & Lois Lapidus Cancer Institute, Sinai Hospital, Baltimore, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25A.1
DOI:  10.1002/0471142727.mb25a01s112
Online Posting Date:  October, 2015
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Abstract

Laser microdissection (LM) offers a relatively rapid and precise method of isolating and removing specified cells from complex tissues for subsequent analysis of their RNA, DNA, protein or metabolite content, thereby allowing assessment of the role of different cell types in the normal physiological or disease processes being studied. In this unit, protocols for the preparation of mammalian frozen tissues, fixed tissues, and cytologic specimens for LM, including tissue freezing, tissue processing and paraffin embedding, histologic sectioning, cell processing, hematoxylin and eosin staining, immunohistochemistry, and image‐guided cell targeting are presented. Also provided are recipes for generating lysis buffers for the recovery of nucleic acids and proteins. The Commentary section addresses the types of specimens that can be utilized for LM and approaches to staining of specimens for cell visualization. Emphasis is placed on the preparation of tissue or cytologic specimens as this is critical to effective LM. © 2015 by John Wiley & Sons, Inc.

Keywords: microdissection; tissues; processing; cytologic samples; isolated cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Frozen Sections for LM
  • Basic Protocol 2: Preparation of Fixed Paraffin‐Embedded Sections
  • Basic Protocol 3: Preparation of Cytologic Specimens for LM: Direct Smears
  • Basic Protocol 4: Preparation of Cytologic Specimens for LM: Cytospin Method
  • Alternate Protocol 1: Removing Blood from Samples for Cytologic Smears or Cytospins
  • Basic Protocol 5: Hematoxylin and Eosin Staining
  • Basic Protocol 6: Immunohistochemistry (IHC) Prior to LM
  • Basic Protocol 7: Expression Microdissection
  • Support Protocol 1: Tissue Fixation and Paraffin Embedding
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Frozen Sections for LM

  Materials
  • Embedding medium (e.g., OCT; Tissue‐Tek)
  • ∼1‐cm maximum‐dimension tissue samples
  • Cryomolds (Tissue‐Tek)
  • Dry‐ice container with lid
  • Aluminum foil
  • Cryostat (e.g., Microm or other vendors), refrigerated to –20°C with tissue platform (chuck) and appropriate blades
  • Glass slides (e.g., Gold Seal plain uncoated slides; Becton Dickinson), membrane slides (e.g., PEN membrane frame slides, Life Technologies), or membrane‐coated slides (e.g., PEN membrane glass slides, Life Technologies)
  • No. 2 pencil or slide marker
  • Slide boxes (optional)
NOTE: All reagents are used and all steps performed at room temperature, unless otherwise specified.

Basic Protocol 2: Preparation of Fixed Paraffin‐Embedded Sections

  Materials
  • Paraffin‐embedded tissue block mounted on appropriate microtome chuck (see protocol 9Support Protocol)
  • Xylenes, histological grade (Sigma‐Aldrich, cat. no. 534056‐4X4L; also available from other vendors)
  • 100%, 95%, and 70% ethanol (prepared using RNase‐free and/or DNase‐free water if the cells are to be microdissected for analysis of their RNA or DNA content)
  • Microtome and appropriate microtome blades (disposable preferred), clean
  • 43° to 44°C water bath
  • Glass histologic slides, plain uncoated, charged, or silanized, or membrane‐coated slides
  • 37° to 42°C oven (optional)
  • Coplin jars or other solvent containers/staining dishes, typically composed of glass [multiple vendors offer dishes and jars designed for use in histology, e.g., Wheaton, item no. 900400 (staining dish) and item no. 900470 (Coplin jar)]
NOTE: All reagents are used and all steps performed at room temperature, unless otherwise specified.

Basic Protocol 3: Preparation of Cytologic Specimens for LM: Direct Smears

  Materials
  • High‐cellularity sample: cellular fluid (e.g., fine‐needle aspiration, suspended cultured cells) or fresh tissue
  • 70% and 95% ethanol (prepared using RNase‐free and/or DNase‐free water if the cells are to be microdissected for analysis of their RNA or DNA content)
  • Hemacytometer cover (optional)
  • Glass slides or membrane‐coated slides, clean
  • Scalpel blade (fresh tissue)
  • Coplin jars or staining dishes [multiple vendors offer dishes and jars designed for use in histology, e.g., Wheaton, item no. 900400 (staining dish) and item no. 900470 (Coplin jar)]
NOTE: All reagents are used and all steps performed at room temperature, unless otherwise specified.

Basic Protocol 4: Preparation of Cytologic Specimens for LM: Cytospin Method

  Materials
  • Low‐cellularity sample such as fine‐needle aspiration or cultured cells suspended in medium
  • 95% and 70% ethanol (prepared using RNase‐free and/or DNase‐free water if the cells are to be microdissected for analysis of their RNA or DNA content)
  • Coplin jars or staining dishes [multiple vendors offer dishes and jars designed for use in histology, e.g., Wheaton, item no. 900400 (staining dish) and item no. 900470 (Coplin jar)]
  • Cytospin instrument and appropriate single sample chamber cytospin device (e.g., Shandon/Lipshaw)
  • Glass or membrane‐coated slides, clean
NOTE: All reagents are used and all steps performed at room temperature, unless otherwise specified.

Alternate Protocol 1: Removing Blood from Samples for Cytologic Smears or Cytospins

  Materials
  • Cytologic sample
  • Sterile saline (i.e., 0.9% w/v NaCl) or balanced salt solution
  • Ficoll‐Paque (GE Healthcare Life Sciences)
  • Centrifuge
  • 50‐ml centrifuge tubes
NOTE: All reagents are used and all steps performed at room temperature, unless otherwise specified.

Basic Protocol 5: Hematoxylin and Eosin Staining

  Materials
  • Sample on a glass slide (see Basic Protocols protocol 11 to protocol 44)
  • 70%, 95%, and 100% ethanol (prepared using RNase‐free and/or DNase‐free water if the cells are to be microdissected for analysis of their RNA or DNA content)
  • Sterile, distilled, or RNase‐free water
  • Mayer's hematoxylin (Thermo Scientific or other vendors); undiluted or diluted 1:5 to 1:10 with water
  • Bluing reagent (Thermo Scientific or other vendors)
  • Eosin Y (Thermo Scientific or other vendors)
  • Xylenes, histological grade (Sigma‐Aldrich, cat. no. 534056‐4X4L; also available from other vendors)
  • Staining rack for microscope slides (VWR International, cat. no. 15153‐821; also available from other vendors)
  • Plastic squeeze bottles for all reagents except xylenes (Nalgene, cat. no. 69103; also available from other vendors)
  • Coplin jar or solvent container/staining dish for xylenes [multiple vendors offer dishes and jars designed for use in histology, e.g., Wheaton, item no. 900400 (staining dish) and item no. 900470 (Coplin jar)]
NOTE: All reagents are used and all steps performed at room temperature, unless otherwise specified.

Basic Protocol 6: Immunohistochemistry (IHC) Prior to LM

  Materials
  • FFPE tissue sample on glass slide ( protocol 2 or protocol 9Support Protocol) or frozen sections ( protocol 1)
  • Xylenes, histological grade (Sigma‐Aldrich, cat. no. 534056‐4X4L; also available from other vendors)
  • 70%, 95%, and 100% ethanol (prepared using RNase‐free and/or DNase‐free water if the cells are to be microdissected for analysis of their RNA or DNA content)
  • Sterile, distilled, RNase‐free or proteinase‐free water
  • Citrate buffer, pH 6.0 (see recipe); antigen retrieval solution can alternatively be purchased from any of multiple vendors (e.g., Biogenex Citra Plus, cat. no. HK080‐9 K)
  • Phosphate‐buffered saline (PBS; e.g., Life Technologies, cat. no. 10010‐023)
  • Peroxidase blocking solution (available from multiple vendors)
  • Primary antibody (mouse or rabbit) to specified antigen
  • Secondary antibody (anti‐mouse or anti‐rabbit) conjugated to horseradish peroxidase (HRP) (available through multiple vendors)
  • 3,3′‐diaminobenzidine (DAB) solution (available through multiple vendors)
  • Coplin jar or solvent container/staining dish for xylenes [multiple vendors offer dishes and jars designed for use in histology, e.g., Wheaton, item no. 900400 (staining dish) and item no. 900470 (Coplin jar)]
  • Heat source: steamer or microwave oven
NOTE: All reagents are used and all steps performed at room temperature, unless otherwise specified.

Basic Protocol 7: Expression Microdissection

  Materials
  • IHC‐stained sample (see protocol 7)
  • Ethylene vinyl acetate (EVA) film (commercially available from multiple vendors, e.g., 3 M)
  • Vacuum‐seal bags (e.g., FoodSaver Pint‐Size Heat‐Seal Bags)
  • Standard vacuum system
  • Blotting paper (extra‐thick; BioRad, cat. no. 170‐3966)
  • SensEpil Flashlamp (Silk'n Home Skinovations, Inc.; http://www.silkn.com)
NOTE: All reagents are used and all steps performed at room temperature, unless otherwise specified.

Support Protocol 1: Tissue Fixation and Paraffin Embedding

  Materials
  • Fresh tissue
  • Fixative of choice (e.g., 70% ethanol)
  • 70%, 80%, 95% and 100% ethanol (prepared using RNase‐free and/or DNase‐free water if the cells are to be microdissected for analysis of their RNA or DNA content)
  • Xylenes, histological grade (Sigma‐Aldrich (cat. no. 534056‐4X4L) or other vendors)
  • Embedding paraffin
  • Tissue cassettes
  • Automated tissue processor (e.g., Leica or Sakura)
  • Scalpel blades (multiple vendors (e.g., Bard‐Parker), size 10 to 23, depending on preference)
  • Embedding molds (e.g., Tissue‐Tek)
  • Embedding center (optional; e.g., Leica)
  • Chuck from microtome that will be used for sectioning in protocol 2
NOTE: All reagents are used and all steps performed at room temperature, unless otherwise specified.
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Figures

Videos

Literature Cited

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Key References
  Legres et al., 2014. See above.
  This article provides an extensive review of different laser microdissection technologies, uses of laser microdissection, and pre‐analytical variables.
  Rodriguez‐Canales et al., 2013. See above.
  This is an extensive review article that covers laser microdissection technology and its uses and provides detailed protocols for tissue preparation and analysis.
Internet Resources
  http://www.niehs.nih.gov/research/resources/protocols/laser/protocols/index.cfm
  This Web site is maintained by the Laboratory of Experimental Pathology of the National Institute of Environmental Health Sciences and is a source of protocols for L.M.
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