Measurement of Expression of the HSP70 Protein Family

Marcelle Bargeron1, Frank R. Sharp2

1 Lilly Research Laboratories, Indianapolis, Indiana, 2 University of Cincinnati Medical Center, Cincinnati, Ohio
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.9
DOI:  10.1002/0471140856.tx0209s07
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The heat shock protein family of proteins include a number of proteins of similar sizes that are inducible by a wide variety of “stress” stimuli including heat shock, ischemia, toxic metals, amino acid analogs, and some chemotherapeutic and anesthetic chemicals. The functions of these proteins are diverse but are generally associated with molecular chaperoning of proteins. This unit contains a wide variety of protocols for analyzing expression of this family at the transcriptional and translational level: northern blot hybridization, in situ hybridization, metabolic labeling and SDS‐PAGE, and immunoblotting.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Quantitative Detection of HSP70 Family mRNA by Northern Blot
  • Basic Protocol 2: Detection of HSP70 Family mRNA on Tissue Sections by in SITU Hybridization
  • Alternate Protocol 1: Emulsion Autoradiography
  • Support Protocol 1: Counterstaining with Cresyl Violet (NISSL)
  • Support Protocol 2: Coverslipping Specimens on Slides
  • Basic Protocol 3: Labeling Stress Proteins with [35S]Methionine
  • Support Protocol 3: Coomassie Blue Staining of Proteins in a Polyacrylamide Gel
  • Basic Protocol 4: Quantitative Detection of HSP70 Family Proteins by Immunoblotting
  • Alternate Protocol 2: Visualization with Chemiluminescent Substrates
  • Support Protocol 4: Reversible Staining of Transferred Protein on Membranes with Ponceau S Solution
  • Basic Protocol 5: Detection of HSP70 Family Proteins in Tissue Sections by Immunohistochemistry
  • Alternate Protocol 3: Nickel‐DAB Intensification of Immunohistochemical Signals
  • Alternate Protocol 4: Double Immunofluorescence Staining for Cell‐Type Identification
  • Alternate Protocol 5: TUNEL Staining of DNA Fragmentation in Combination with Immunohistochemistry
  • Support Protocol 5: Preparing Gelatin‐Coated Slides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Quantitative Detection of HSP70 Family mRNA by Northern Blot

  Materials
  • Animals subjected to a stressful stimulus (Table 2.9.1)
  • Anesthesia cocktail (see recipe)
  • TRIzol reagent (Life Technologies; TRIzol Reagent, Sigma; or RNA STAT‐60, Tel Test)
  • 5:1 (v/v) acid phenol/chloroform solution, pH 4.7 (Ambion)
  • Horizontal formaldehyde/1% (w/v) agarose gel ( appendix 3E)
  • Nylon membrane, positively charged (e.g., Hybond, Amersham; Nytran SuPerCharge, Schleicher and Schuell; BrightStar‐Plus, Ambion)
  • 0.02% (w/v) methylene blue staining solution (see recipe)
  • 2× and 20× SSC (see recipe)
  • 32P‐labeled antisense oligonucleotide probe (Table 2.9.2) and sense control probe, (single‐stranded, unlabeled oligonucleotides are obtained from Oligos Etc., or other commercial source)
  • Prehybridization solution (for recipe, see appendix 3E, Basic Protocol, step 25)
  • 50% (w/v) dextran sulfate (see recipe)
  • 2× and 1× SSPE (see recipe)
  • 20% (w/v) SDS
  • UV cross‐linker (Stratagene)
  • Sealable bag or glass hybridization bottle
  • Hybridization oven or 42°C shaker water bath
  • Additional reagents and equipment for RNA extraction, agarose gel electrophoresis ( appendix 3A), oligonucleotide labeling ( appendix 3E), and northern blot hybridization ( appendix 3E)
NOTE: To protect from RNase contamination and fingerprint smudges, always wear clean, powder‐free gloves when working RNA. Do not reuse gloves.

Basic Protocol 2: Detection of HSP70 Family mRNA on Tissue Sections by in SITU Hybridization

  Materials
  • Animals subjected to a stressful stimulus (Table 2.9.1)
  • Anesthesia cocktail (see recipe)
  • 5 M DTT (see recipe)
  • [35S]‐ or [33P]‐labeled antisense oligonucleotide probe (Table 2.9.2) and sense control probe (single‐stranded, unlabeled oligonucleotides are obtained from Oligo Etc. or other commercial source)
  • In situ hybridization cocktail (see recipe)
  • 10 mg/ml heat‐denatured salmon sperm DNA stock
  • 1× SSC (see recipe), sterile, 55°C
  • H 2O, deionized, sterile
  • 75%, 95%, and 100% ethanol
  • Dissecting instruments
  • Isopentane on powdered dry ice bath
  • Cryostat
  • Precoated slides with painted corners (e.g., ProbeOn or Superfrost Plus, Fisher Scientific)
  • Moist chamber with cover
  • Parafilm
  • Slide rack
  • Staining dish in a 55°C water bath
  • Hair dryer
  • Autoradiographic cassette
  • X‐ray film (Kodak SB5 for manual processing or BioMax MR one sided emulsion for automated processor)
  • Additional reagents and equipment for oligonucleotide labeling ( appendix 3E)
NOTE: To protect from RNase contamination and fingerprint smudges, always wear clean, powder‐free gloves when working RNA. Do not reuse gloves.

Alternate Protocol 1: Emulsion Autoradiography

  Materials
  • NTB‐2 autoradiographic emulsion (Kodak)
  • Slides with radioactively hybridized sections (see protocol 2, step )
  • D19 or Dektol developer and fixer (Kodak), freshly made
  • Milli‐Q‐purified water
  • 43°C water bath
  • Black light‐tight slide boxes
  • Dessicant packs (Drierite)
  • Dipping container
  • Forceps with blunt ends (optional)
  • Slide rack
  • Slide dryer (optional)
  • Black electrical tape
  • Staining dishes
  • Light microscope
NOTE: Use very clean glassware and reagent‐grade solutions. Wear powder‐free gloves for all procedures.CAUTION: All procedures involving autoradiographic emulsion should be performed in complete darkness or under safelight conditions. The autoradiography emulsion must be refrigerated at 4°C until use. Because it is in a solid form at 4° to 10°C, the emulsion must be liquified by heating to ∼45°C before use. Do not freeze or store the emulsion at room temperature to avoid high background fog and loss of detection ability.

Support Protocol 1: Counterstaining with Cresyl Violet (NISSL)

  Materials
  • Dry tissue specimen on slides from immunohistochemistry or in situ hybridization
  • Chloroform (optional)
  • Ether (optional)
  • 70%, 95%, and 100% ethanol
  • Milli‐Q‐purified water
  • Cresyl violet staining solution (see recipe)
  • Glacial acetic acid
  • Slide racks
  • Glass staining dishes
NOTE: Perform all steps at room temperature, in a well‐ventilated fume hood. Wear gloves at all times.

Support Protocol 2: Coverslipping Specimens on Slides

  Materials
  • Tissue specimens mounted and dried on slides
  • 70%, 95%, and 100% ethanol
  • Clearing solution such as xylene or Hemo‐D (Fisher)
  • Mounting medium (e.g., DePeX, BDH; Permount, Fisher; or equivalent)
  • Slide racks
  • Glass staining dishes
  • Forceps with blunt end
  • Glass coverslips, large enough to cover the tissue specimen
CAUTION: Xylene is a toxic solvent and all steps of counterstaining and coverslipping should be carried out in a fume hood or ventilated table.

Basic Protocol 3: Labeling Stress Proteins with [35S]Methionine

  Materials
  • Tissue culture cells in 35‐ to 100‐mm plates
  • Methionine‐free medium for cell culture
  • stressful stimulus (see Tables 2.9.1 and 2.9.3)
  • 1000 Ci/mmol [35S]methionine (Amersham Pharmacia Biotech or NEN)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold
  • 1× SDS sample buffer (see recipe)
  • Protein quantitation kit (Pierce or Bio‐Rad)
  • Bovine serum albumin (BSA) protein standards
  • Molecular weight markers
  • 2‐mercaptoethanol
  • 1% (w/v) bromphenol blue (see recipe)
  • Water‐based scintillant solution
  • 1.5‐ml microcentrifuge tubes
  • Cell scraper
  • 100°C water bath
  • Filter‐membrane spin column (optional; Amicon or Sigma)
  • Additional reagents and equipment for SDS‐PAGE ( appendix 3A), Coomassie blue staining of gels (see protocol 7), and gel drying and autoradiography ( appendix 3D)

Support Protocol 3: Coomassie Blue Staining of Proteins in a Polyacrylamide Gel

  Materials
  • SDS‐PAGE gel from separation of stress proteins including the HSP70 proteins (see protocol 6, step )
  • Gel fixing solution (see recipe)
  • Coomassie blue staining solution (see recipe)
  • Gel destaining solution (see recipe)
  • Clean glass or plastic dishes slightly larger than the gel with lid or plastic wrap
  • Platform shaker
  • Foam sponge (optional)

Basic Protocol 4: Quantitative Detection of HSP70 Family Proteins by Immunoblotting

  Materials
  • Cell culture or animals subjected to a stressful stimulus
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1× SDS sample buffer (see recipe)
  • Protein quantification kit (Pierce or Bio‐Rad)
  • Bovine serum albumin (BSA) standards
  • Molecular weight markers
  • 2‐mercaptoethanol
  • 1% (w/v) bromphenol blue (see recipe)
  • 0.2‐µm mesh nitrocellulose or polyvinylidine difluoride (PVDF) membrane (Biorad, Scleicher and Schuell, NOVEX)
  • Immunoblot blocking solution (see recipe)
  • Immunoblot wash solution (see recipe)
  • Primary antibodies against the HSP70 protein family (see Table 2.9.4)
  • Secondary antibody, biotinylated or conjugated to horseradish peroxidase (Amersham Pharmacia Biotech or Vector)
  • 0.1 M phosphate buffer, pH 7.4 (see recipe)
  • Standard Elite Vectastain ABC kit (Vector)
  • 10‐mg tablets of 3,3′‐diaminobenzidine tetrachloride (DAB; Sigma)
  • 30% hydrogen peroxide ≤1 to 2 months old
  • 100°C water bath
  • Rotating platform shaker
  • Additional reagents and equipment for SDS‐PAGE ( appendix 3A), transfer of proteins to membrane (unit 2.3), and Ponceau S staining (see protocol 10)

Alternate Protocol 2: Visualization with Chemiluminescent Substrates

  Materials
  • Chemiluminescence horseradish peroxidase (HRP) substrate kit (e.g., ECL, Amersham Pharmacia Biotech or equivalent)
  • Immunoblot probed with secondary antibody directly conjugated to horseradish peroxidase (see protocol 8, step )
  • 0.1 M phosphate buffer, pH 7.4 (see recipe)
  • 50‐ml conical plastic centrifuge tubes
  • Rotating platform shaker
  • X‐ray film (e.g., X‐Omat AR‐5, Kodak)
  • Automatic X‐ray film processor

Support Protocol 4: Reversible Staining of Transferred Protein on Membranes with Ponceau S Solution

  Materials
  • Membrane with transferred proteins (see protocol 8, step )
  • Ponceau S staining solution (see recipe)

Basic Protocol 5: Detection of HSP70 Family Proteins in Tissue Sections by Immunohistochemistry

  Materials
  • Animals or cells subjected to a stressful stimulus (see Tables 2.9.1 and 2.9.3)
  • Anesthesia cocktail (see recipe)
  • 0.9% (w/v) sodium chloride (saline) solution, ice cold
  • 4% (w/v) paraformaldehyde working solution (PFA; see recipe), ice cold
  • 30% (w/v) sucrose (see recipe)
  • 0.1 M and 0.05 M phosphate buffer, pH 7.4 (PB; see recipe)
  • Immunohistochemistry peroxidase inhibiting solution (optional, see recipe)
  • Immunohistochemistry blocking solution (see recipe)
  • Primary antibodies against the HSP70 protein family (see Table 2.9.4)
  • Immunohistochemistry antibody dilution buffer (see recipe)
  • Secondary antibody conjugated to biotin
  • Avidin/peroxidase complex kit (e.g., ABC Standard Elite Vectastain kit, Vector)
  • 10‐mg tablets of 3,3′‐diaminobenzidine tetrachloride (DAB, Sigma)
  • 30% (v/v) hydrogen peroxide solution, stabilized, ≤1 to 2 months old (Sigma)
  • Vibratome or freezing microtome
  • Paintbrush with small, fine end
  • 6‐ or 12‐well tissue culture plates
  • Rotating platform shaker
  • Gelatin‐coated histology slides (see protocol 15)
  • Additional reagents and equipment for preparing gelatin‐coated slides (see protocol 15) and coverslipping slides (see protocol 5)

Alternate Protocol 3: Nickel‐DAB Intensification of Immunohistochemical Signals

  • 0.175 M sodium acetate, pH 6.7 (see recipe)
  • Nickel sulfate

Alternate Protocol 4: Double Immunofluorescence Staining for Cell‐Type Identification

  • Two primary antibodies made in different species
  • Secondary antibodies: one conjugated to biotin, one conjugated to a fluorochrome
  • Avidin‐conjugated fluorochrome
  • Fluorescence mounting medium: 3:1 (v/v) glycerol/0.1 M phosphate buffer, pH 7.4, containing 0.1% (w/v) paraphenylenediamine or Fluoromount‐G (Southern Biotech) or Vectashield (Vector) or Slowfade‐Light Antifade (Molecular Probes)
  • Light‐proof box

Alternate Protocol 5: TUNEL Staining of DNA Fragmentation in Combination with Immunohistochemistry

  • 0.3% (v/v) Triton X‐100 in 0.1 M phosphate buffer, pH 7.4 (see recipe for buffer)
  • 20 µg/ml proteinase K in 0.1 M phosphate buffer, pH 7.4 (see recipe for buffer; optional)
  • 1× and 5× TdT buffer (Gibco BRL or Roche Molecular Biochemicals)
  • 400 µM biotinylated 14‐dATP (Life Technologies)
  • 15 U/µl TdT (terminal deoxynucleotidyl transferase; Gibco BRL or Roche Molecular Biochemicals)
  • 2× SSC (optional; see recipe)
  • 2% (w/v) bovine serum albumin (BSA; see recipe; optional)
  • 0.175 M sodium acetate, pH 6.7 (see recipe)
  • Nickel sulfate
  • 75%, 95%, and 100% ethanol
  • Powdered dry ice
  • Cryostat
  • Gelatin‐coated histology slides (see protocol 15) or precoated slides (e.g., SuperfrostPlus or Probe‐On, Fisher)
  • PAP pen or equivalent hydrophobic pen (ImmEdge Pen, Vector or PAP Pen, ScyTek)
  • Humidified chamber with lid
  • Coplin staining jars
  • Light microscope

Support Protocol 5: Preparing Gelatin‐Coated Slides

  Materials
  • Milli‐Q‐purified water
  • 1% (v/v) hydrochloric acid solution
  • 70% and 95% ethanol
  • Gelatin (type Bloom 275)
  • Chromium potassium sulfate
  • Slide racks
  • 25 × 75–mm microslides with a frosted end, precleaned (Fisher)
  • Glass staining dishes
  • Whatman 3MM filter paper
  • Storage slide boxes
NOTE: To avoid fingerprint smudges, always wear clean, powder‐free gloves when manipulating the slides. Wear safety goggles and gloves to avoid acid burn.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bergeron, M., Mivechi, N.F., Giaccia, A.M., and Giffard, R.G. 1996. Mechanism of heat shock protein 72 induction in primary cultured astrocytes after oxygen‐glucose deprivation. Neurol. Res. 18:64‐72.
   Kinouchi, H., Sharp, F.R., Hill, M.P., Koistinaho, J., Sagar, S.M., and Chan, P.K. 1993. Induction of 70‐kDa heat shock protein and hsp70 mRNA following transient focal ischemia in the rat. J. Cerebr. Blood Flow Metab. 13:105‐115.
   Longo, F.M., Wang, P., Narasimhan, P., Zhang, J.S., Chen, J., Massa, S.M., and Sharp, F.R. 1993. cDNA cloning and expression of stress‐induced rat hsp70 in normal and injured rat brain. J. Neurosci. Res. 36:325‐335.
   Massa, S.M., Longo, F.M., Zuo, J., Wang, S., Chen, J., and Sharp, F.R. 1995. Cloning of rat grp75, an hsp70‐family member, and its expression in normal and ischemic brain. J. Neurosci. Res. 40:807‐819.
   Massa, S.M., Swanson, R.A., and Sharp, F.R. 1996. The stress gene response in brain Cerebrovasc. Brain Metab. Rev. 8:95‐158.
   Miller, E.K., Raese, J.D., and Morrison‐Bogorad, M. 1991. Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment. J. Neurochem. 56:2060‐2071.
   Narasimhan, P., Swanson, R.A., Sagar, S.M., and Sharp, F.R. 1996. Astrocyte survival and HSP70 heat shock protein induction following heat shock and acidosis. Glia 17:147‐159.
   Pardue, M.L. 1985. In situ hydridization. In Nucleic Acid Hybridization: A Practical Approach (B.D. Hanes and S.J. Higgins, eds.) pp. 179‐202. IRL Press, Oxford, England.
   Parsell, D.A. and Lindquist, S. 1994. Heat shock proteins and stress tolerance. In The Biology of Heat Shock Proteins and Molecular Chaperones (R.I. Morimoto, A. Tissières, and C. Georgopoulos, eds.) pp. 457‐494. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Sharp, F.R., Massa, S.M., and Swanson, R.A. 1999. Heat‐shock protein protection. Trends Neurosci. 22:97‐99.
   Stahl, W.L., Eakin, T.J., and Baskin, D.G. 1993. Selection of oligonucleotide probes for detection of mRNA isoforms. J. Histochem. Cytochem. 41:1735‐1740.
   Welch, W.J. 1990. The mammalian stress response: Cell physiology and biochemistry of stress proteins. In Stress Proteins in Biology and Medicine. (R.I. Morimoto, A. Tissières, and C. Georgopoulos, eds.) pp. 223‐278. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Welch, W.J. 1992. Mammalian stress response: Cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol. Rev. 72:1063‐1081.
   Welch, W.J. and Suhan, J.P. 1986. Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J. Cell. Biol. 103:2035‐2052.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library