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Immunohistochemical Detection of S-Nitrosylated Proteins

  • Andrew J. Gow
  • Christiana W. Davis
  • David Munson
  • Harry Ischiropoulos
Part of the Methods in Molecular Biology™ book series (MIMB, volume 279)

Abstract

Accumulating evidence shows that S-nitrosothiols, formed by the addition of nitric oxide (NO) to a cysteine thiol, S-nitrosylation, are involved in basal cellular regulation. It has been proposed that SNO formation/removal may be disrupted in a variety of pathophysiological conditions. Two types of methodology are presently available to identify specific S-nitrosylated proteins: (1) derivatization and (2) post-purification chemical detection. Neither of these techniques allows for in situ visualization of SNOs. Recently, we demonstrated that an antibody generated to the SNO moiety could be used to detect SNO formation from each of three isoforms of NOS by immunohistochemistry. This chapter details the immunohistochemical methodology used to detect SNOs in situ, offering a potentially powerful alternative for detection of SNO within tissue sections.

Key Words

S-Nitrosothiols immunohistochemistry nitric oxide synthase nitrosylation 

1 Introduction

In the increasingly complex biology of NO-related signaling, the formation of S-nitrosothiols (SNOs) is recognized as one potential pathway for NO-based cellular regulation (1). A number of proteins with diverse functions have been identified as S-nitrosylated in vivo including the NMDA receptor (2), p21ras3, OxyR (4), necrosis factor (NF-κB) (5), caspase-3 (6). At present, there are two types of methodology available to identify specific S-nitrosylated proteins, namely isolation and chemical analysis (7) or derivatization followed by electrophoretic separation (8). The identification of specific protein S-nitrosylation in situ has been difficult to achieve largely because of a variety of technical difficulties. Moreover, it has been difficult to demonstrate that SNO-adduct formation was associated with in vivo activation of nitric oxide synthase (NOS) isoforms. Recently, it has been demonstrated that an antibody generated against conjugated SNO can be used for detection of SNO proteins by enzyme-linked immunosorbent assay (ELISA), electrophoresis, and immunohistochemistry (9). Furthermore, these studies showed that each isoform of NOS is capable of generating SNO upon stimulation. There now exists a challenge to utilize this immunological approach to examine the production of SNO in a variety of pathophysiological states in which NOS function is altered. Here, we present an immunohistochemical method, utilizing anti-SNO antibodies to examine SNO production in tissue sections.

Three pathways for the production of SNO within biological systems have been proposed, namely nitrosation via the formation of N2O3 (10), metal catalysis (11,12), and direct reaction followed by electron abstraction (13). The first of these operates through the formation of the nitrosonium cation (NO+), which reacts readily with nucleophilic targets, such as thiol. There is some debate as to the possibility of nitrosonium formation under physiological conditions, although under conditions of combined oxidative and nitrosative stress, such reactions will become more likely. The precise mechanisms involved in these reactions are unclear, either there is a concerted electron abstraction and thiol reaction as appears to operate in cerulo plasmin (11) or they may proceed through a radical intermediate (R-S-NOH). SNO formation within hemoglobin has been shown to be associated with the formation of a novel radical intermediate (14).

The nitrosonium chemistry can be capitalized upon to make SNO-positive controls. By mixing equimolar quantities of acid and nitrite, one can generate nitrosonium intermediates. The addition of these intermediates to tissue sections ( Subheading 3.2. , step 4) results in the generation of SNO adducts within these tissues. In addition, this chemistry can be utilized to generate SNO equivalents within solution. Routinely, we synthesize S-nitrosoglutathione (GSNO) by mixing acidified glutathione with nitrite in equimolar quantities (see Note 1 ). Importantly, just as metals can be involved in SNO synthesis, they are readily able to catalyze the breakdown of the SNO bond. In particular, copper and mercury ions are known to operate in this regard. Indeed, these reactions have been utilized to construct chemical assays for SNO (the Saville and copper/cysteine assays [15]). Mercury is unique in its reaction with SNO among metals; rather than merely catalyzing the cleavage of the bond, mercury forms a covalent bond with the sulfur (a mercaptan). This is critically important for the use of mercury as a negative control within SNO immunohistochemistry, as it means that once an SNO moiety has reacted with the mercury compound, the thiol is captured in the form of the mercaptan.

2 Materials

  1. 1.

    Phosphate-buffered saline (PBS): 2.88 g Na2HPO4, 16.0 g NaCl, 0.4 g KCl, and 0.48 g KH2PO4 in 2000 mL of water, pH to 7.2.

     
  2. 2.

    PBS/Triton: 3 mL of Triton X-100 in 1 L of PBS to make a 0.3% Triton-PBS mixtures.

     
  3. 3.

    Blocking buffer: 100% goat serum.

     
  4. 4.

    Borohydride: 0.1 g of NaBH4 in 100 mL PBS (see Note 2 ).

     
  5. 5.

    Antigen retrieval solution: 0.28 g of EDTA and 0.41 g of Tris base in 750 mL of water (pH to anywhere within the range 6.0–9.0; as is appropriate for the tissue with which one is working).

     
  6. 6.

    Nitrosation solution: 0.34 g NaNO2 in 2 mL of 0.1M HCl (see Note 3 ).

     
  7. 7.

    Organic mercury: 0.019 g p-hydroxymercuricbenzoatesulfone (pHMB) in 15 mL PBS (see Note 4 ).

     
  8. 8.

    Primary antibody: Antinitrosocysteine monoclonal antibody (in addition a rabbit polyclonal is available and this has also been used in this protocol) obtained from A. G. Scientific (www.agscientific.com; product no. N-1078). Supplied as a lyophilized powder, from PBS, and can be reconstituted with 150 μL distilled water, aliquoted and frozen at −20°C. Aliquots can be frozen and thawed up to five times.

     
  9. 9.

    Secondary antibody: Anti-mouse F(ab)2 fragments conjugated with fluorescein isothiocyanate (FITC) for immunofluorescence or horseradish peroxidase (HRP) for 3,3′-diaminobenzidine (DAB) staining (Sigma). 1:100 dilution in 100% goat serum is optimal for both fluorescent and DAB staining.

     
  10. 10.

    Hydrogen peroxide: 5 mL of H2O2 in 25 mL methanol.

     
  11. 11.

    Primary antibody solution: Add 10 μL of reconstituted primary antibody to the blocking buffer (100% goat serum) to produce a 1:100 dilution that is optimal for immunohistochemistry (see Note 5 ).

     
  12. 12.

    PBS/BSA: 0.1g of bovine serum albumin (BSA) (fatty-acid-free fraction V) in 100 mL PBS (store at 4°C).

     
  13. 13.

    DAB solution: 5 mg DAB in 10 mL of PBS containing 0.1% BSA (fatty-acid-free fraction V). This solution should be filtered prior to use.

     

3 Methods

3.1 Immunofluorescent Detection of Tissue SNO

  1. 1.

    Both frozen and paraffinized tissue sections can be used for analysis. As both negative and positive controls are critical to the assessment of the degree of staining, it is preferable to have three serial sections mounted per slide.

     
  2. 2.

    Paraffinized tissue must be deparaffinized by placing slides within Coplin jars for 3 min in a succession of solutions, namely 2X xylene, 100% ethanol, 70% ethanol, 30% ethanol, and then distilled water.

     
  3. 3.

    Following deparaffinization or defrosting, slides are washed two times in PBS/Triton for 5 min each.

     
  4. 4.

    Slides are placed in antigen retrieval solution for 5 min. After incubation, the slides are microwaved twice for 5 min with a 1-min interval at full power. If necessary, more antigen retrieval solution can be added (see Note 6 ).

     
  5. 5.

    Wash slides for 5 min in distilled water and then 5 min in PBS/Triton.

     
  6. 6.

    After washing, encircle sections with a hydrophobic pen.

     
  7. 7.

    At this point, the slides are ready for staining and positive and negative controls can be created (see Subheading 3.2. ).

     
  8. 8.

    Wash slides twice for 5 min in PBS/Triton.

     
  9. 9.

    Slides are immersed for 2 min three times in freshly prepared borohydride to remove background fluorescence (see Note 7 ).

     
  10. 10.

    Incubate slides for 30 min in blocking buffer (see Note 8 ).

     
  11. 11.

    Incubate slides overnight in the dark at 4°C in primary antibody (see Note 9 ).

     
  12. 12.

    Wash slides twice for 5 min in PBS/Triton.

     
  13. 13.

    Incubate slides with secondary antibody solution at room temperature in the dark for 2 h.

     
  14. 14.

    Wash slides twice for 5 min in PBS/Triton.

     
  15. 15.

    Wash slides twice for 5 min in PBS.

     
  16. 16.

    Wash in distilled water for 2 min.

     
  17. 17.

    Allow to air-dry in the dark.

     
  18. 18.

    Place a cover slip over the sections. Mounting solutions can be used, although it is preferred to use none and examine the slides immediately.

     

3.2 Immunohistochemistry Controls

  1. 1.

    Tissue staining with antigen-competed primary antibody. One potential negative control to gage the specificity of the staining achieved is to utilize antigen competition. For this control, 0.5 mM of GSNO is added to the primary antibody solution (see Note 1 ). The primary antibody solution is incubated for 2 h at room temperature prior to incubation with the slides.

     
  2. 2.

    Conversion of SNO to a mercaptan with mercury solution. As stated previously, the S-nitroso bond can be specifically broken by the reaction of sulfur with mercury to form a mercaptan. This can be achieved using either inorganic mercury, namely HgCl2, or an organically linked mercurial such as pHMB. In our experience, although chemically inorganic mercury is more effective at SNO cleavage, pHMB works best in staining. Therefore, for a negative control, we utilize a 30-min incubation with organic mercury ( Subheading 3.1. , step 7). The advantage of this step is that it controls directly for the specificity of the interaction of the antibody with SNO. Its disadvantage is that one cannot be certain of the degree of SNO cleavage that has been achieved with the use of the mercurial compound.

     
  3. 3.

    Tissue staining with nonspecific purified mouse IgG. An alternative negative control can be used of replacing the primary antibody with a similar titer of nonspecific mouse IgG.

     
  4. 4.

    Generation of positive slides by acidified nitrite treatment. It is beneficial to perform positive controls in order to check the quality of the stain. Positive controls are generated by placing fresh nitrosation solution on the tissue section and incubating for 30 min at room temperature at (see Subheading 3.1. , step 7).

     

3.3 DAB Staining for SNO

Adaptations of the protocol to conduct nonfluorescent staining for SNO utilizing DAB.

  1. 1.

    At step 9 of Subheading 3.1. , replace immersion in borohydride with a 20-min incubation with hydrogen peroxide.

     
  2. 2.

    Wash two times for 5 min in PBS/BSA.

     
  3. 3.

    Incubate with HRP-linked F(ab)2 secondary for 1 h

     
  4. 4.

    Wash twice with PBS/BSA for 5 min.

     
  5. 5.

    Add 10 μL of 30% H2O2 solution to DAB solution (mix well) and apply to the tissue sections.

     
  6. 6.

    Wait until the sections start to turn brown (pay particular attention to the positive controls).

     
  7. 7.

    Stop the reaction by rinsing the slides with PBS and then distilled water.

     
  8. 8.

    For counterstaining, follow steps in succession: 3X in xylene, 3 min each; 3X in 100% ethanol for 3 min; 2X in 95% ethanol for 2 min; several dips in water; 1 min in hematoxylin; several dips in water; 10 s in bluing agent; several dips in water; 3X in 95% ethanol for 2 min; 3X in 100% ethanol for 3 min; 3X in xylene for 3 min (see Note 10 ).

     
  9. 9.

    Air-dry in dark.

     
  10. 10.

    Mount with xylene base media and cover slip.

     

4 Notes

  1. 1.

    S-Nitrosoglutathione can be synthesized by mixing 0.5M glutathione dissolved in 0.5MHCl containing 0.1 μM diethylenetriamine pentaacetic acid (DTPA) with 0.5M NaNO2 dissolved in water containing DTPA. This makes a 0.25M GSNO solution that can be diluted to 0.05M in antibody solution and then further diluted in the primary antibody solution.

     
  2. 2.

    The borohydride solution should be made up fresh and immediately before use.

     
  3. 3.

    Upon mixing the nitrite and HCl nitrosonium, ions are formed immediately. If left on the bench for any period of time, these ions are lost and nitrogen equivalents are lost to the gas phase in the form of NO and NO2. Therefore, these two reactants are mixed immediately prior to application to the tissues.

     
  4. 4.

    This solution should be stored in the dark.

     
  5. 5.

    The antibody dilution listed is optimal for most tissues. However, in any individual case, the optimal signal to background staining should be determined by serial dilution.

     
  6. 6.

    Frozen sections are considerably more fragile than paraffin embedded. Therefore, considerable care should be taken in their handling. The antigen retrieval step should only be used for paraffin sections, as it damages frozen ones and does not appear to improve staining in these sections.

     
  7. 7.

    Routinely with paraffin sections, slides are immersed in the borohydride solution, with each change of solution being made up immediately before use. Agitation of the sections during the washes has resulted in more efficient background reduction. When using frozen sections, we have found that this sort of treatment can cause damage to the tissues. Therefore, we spot apply and tap off fresh borohydride rather than immerse the whole slide.

     
  8. 8.

    Glycine can be added to the blocking step in order to block reactive aldehydes if background fluorescence persists.

     
  9. 9.

    All incubations of slides are performed in humidified boxes.

     
  10. 10.

    All alcohols used in counterstaining must be changed after each use. Using the same solution twice affects the degree of staining. Do not use the same alcohol solutions used for deparaffinizing.

     

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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Andrew J. Gow
    • 1
  • Christiana W. Davis
    • 2
  • David Munson
    • 2
  • Harry Ischiropoulos
    • 3
    • 4
  1. 1.Division of Neonatology, Joseph Stokes Junior Research Institute, and Abramson Research CenterChildren’s Hospital of PhiladelphiaPhiladelphia
  2. 2.Division of NeonatologyChildren’s Hospital of PhiladelphiaPhiladelphia
  3. 3.Division of Neonatology, Joseph Stokes Junior Research Institute, and Abramson Research CenterChildren’s Hospital of PhiladelphiaPhiladelphia
  4. 4.Department of Biochemistry and BiophysicsUniversity of Pennsylvania School of MedicinePhiladelphia

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