Histochemical Methods for the Detection of Apoptosis in the Nervous System

Tinmarla Frances Oo1, Robert E. Burke1

1 Columbia University Medical Center, New York, New York
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.15
DOI:  10.1002/0471142301.ns0115s39
Online Posting Date:  April, 2007
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Abstract

Neuroscientists often need to detect neuron death at the light microscope level in tissue sections derived from animal models of neurological disease. In many instances there is a need to detect apoptosis, the most common morphology of programmed cell death. This unit provides two protocols for the detection of apoptosis by immunostaining for either activated forms of caspases or their cleavage products. When used in conjunction with nuclear dyes, these protocols permit visualization not only of caspase activation, but also the nuclear chromatin clumps characteristic of apoptosis. The first protocol utilizes peroxidaseÔÇÉmediated chromogen deposition to visualize antibodies by brightfield microscopy. The second protocol utilizes fluorophores to visualize antibodies by epifluorescence. Double immunofluorescence labeling can be performed to identify the phenotype of cells in which caspases are activated. Not all cell death is apoptotic. Therefore, a third protocol is presented for suppressed silver staining, a useful method to screen for all morphologic forms of cell death.

Keywords: apoptosis; programmed cell death; caspases; immunohistochemistry; silver staining

     
 
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Table of Contents

  • Basic Protocol 1: Immunoperoxidase Labeling of Activated Caspases or Their Cleavage Products
  • Basic Protocol 2: Immunofluorescence Double‐Labeling of Activated Caspases or Their Cleavage Products and Cellular Antigens
  • Basic Protocol 3: Suppressed Silver Staining of Brain Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunoperoxidase Labeling of Activated Caspases or Their Cleavage Products

  Materials
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.1, 4°C
  • Fixed brain tissue sections (20‐ to 30‐µm)
  • 0.1 M phosphate‐buffered saline/0.5% (w/v) bovine serum albumin (PBS/BSA; see recipe)
  • 0.1 M phosphate‐buffered saline/0.5% (w/v) bovine serum albumin/0.1% (v/v) Triton X‐100 (PBS/BSA‐T; see recipe)
  • Primary rabbit anti‐caspase or anti‐caspase cleavage product antibodies
  • Biotinylated protein A (Cowan strain; see recipe)
  • Vectastain ABC peroxidase kit (Vector Labs) containing:
    • Reagent A (avidin)
    • Reagent B (biotinylated horseradish peroxidase)
  • Diaminobenzidine tetrahydrochloride hydrate (DAB; Aldrich, no. 261890)
  • 0.1 M Tris buffer (see recipe)
  • Glucose oxidase (see recipe)
  • Ammonium chloride (see recipe)
  • D‐(+)‐glucose (see recipe)
  • Chloroform solution (see recipe)
  • 95% (v/v) and 100% (v/v) ethanol
  • Xylenes
  • Thionin working solution (see recipe)
  • Formalin acetic acid solution (see recipe)
  • 1‐Butanol (Fisher, A‐400)
  • Cedar wood oil (Fisher, O‐40)
  • Permount (Fisher, SP‐15)
  • 6‐ and 12‐well tissue culture plates
  • Red sable brushes for transferring tissue sections (Ted Pella)
  • Platform shaker
  • 150‐ml filter units (0.22‐µm filter) (Nalgene)
  • 115‐ml small staining dish (Thomas)
  • Superfrost Plus microscope slides (Fisher)
  • Coverslips (Fisher, 22 × 50)
  • Tissue–Tek staining outfit with vertical slide holder (Thomas)
  • Dissecting microscope (Nikon)

Basic Protocol 2: Immunofluorescence Double‐Labeling of Activated Caspases or Their Cleavage Products and Cellular Antigens

  Materials
  • Phosphate‐buffered saline (PBS; see recipe)
  • Fixed brain tissue sections (20‐ to 30‐µm)
  • Normal goat serum (Vector Labs, Catalog number S‐1000)
  • Normal horse serum (Vector Labs, Catalog number S‐2000)
  • Primary antibodies:
    • Mouse monoclonal antibody
    • Rabbit polyclonal antibody
  • Primary antibody diluent (see recipe)
  • Tris‐buffered saline (TBS; see recipe)
  • Secondary antibody diluent (see recipe)
  • Secondary antibodies:
    • Texas red‐conjugated horse‐anti‐mouse (Vector, TI‐2000)
    • Fluorescein‐conjugated goat‐anti‐rabbit (Vector, FI‐1000)
  • 1:500 Hoechst working solution (see recipe)
  • Antifade medium (Dako, S‐3023)
  • 12‐well tissue culture plates
  • Red sable brushes for transferring tissue sections (Ted Pella)
  • Platform shaker
  • Mounting dish
  • Superfrost Plus microscope slides (Fisher)
  • 22 × 50–mm coverslips
  • Black plastic slide boxes (VWR)

Basic Protocol 3: Suppressed Silver Staining of Brain Sections

  Materials
  • 0.2 N hydrochloric acid
  • Chromium potassium sulfate in gelatin subbing solution (see recipe)
  • Brain tissue (30‐µm sections)
  • 4% paraformaldehyde in 0.1 M phosphate buffer (PF/PB; see recipe), 4°C
  • Pretreating solution (see recipe)
  • Impregnating solution (see recipe)
  • Washing solution (see recipe)
  • Developing solution (see recipe)
  • Formalin acetic acid solution (see recipe)
  • 70%, 95%, 100% ethanol
  • 1‐Butanol (Fisher)
  • Xylenes (Fisher)
  • Permount (Fisher)
  • Microscope slides (Fisher)
  • Synthetic brushes
  • Tissue basket with polyamide nylon fiber bottom (Tetko; see recipe)
  • Large and small staining dish outfit with cover (Thomas)
  • Glass slide holders with carrier (Thomas)
  • Platform shaker (VWR)
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Figures

Videos

Literature Cited

Literature Cited
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