Contents

• Required Reagents and Equipment
• Buffer Preparation
• DNA In Situ Hybridization Procedure (without "CARD" System)
• Protocol for Nanogold®-Silver In Situ Hybridization Kit with "CARD" System
  1. Using NEN Biotinylated Tyramides
  2. Using Dako Biotinylated Tyramides
• RNA In Situ Hybridization Procedure (with "CARD" System)
• Notes: Buffer formulations
• References

Advantages

1. More sensitive than NBT-BCIP alkaline phosphatase or peroxidase in combination with label amplification single gene copy resolution.
2. No need for the more lengthy PCR procedures for most cases.
3. Avoid false positives found with PCR due to mispriming and amplicon diffusion.
4. Black color easily seen with standard bright field microscope; no expensive fluorescent optics required, or problems with autofluorescence and loss of signal from bleaching.
5. Black color compatible with full strength standard cell and nuclear stains (H & E, nuclear fast red, methyl green) which greatly improves morphological assessment.
6. May be used for EM studies as well.

The heart of this new technological improvement is Nanogold®-Streptavidin (NG-SA), a small 1.4 nm gold nanoparticle cluster covalently attached to SA, which has excellent tissue and nuclear penetration. An optimized protocol allows very low or even single gene copy sensitivity. A brief silver development step gives a specific and permanent black signal at the targeted probe. If additional sensitivity is required, a biotin amplification step may be added using "CARD" (i.e. CAtalyzed Reporter Deposition)³ which is also known as "TSA" (i.e. Tyramide Signal Amplification). With this adjunct NG-SA routinely gives single copy resolution without PCR.

Reagents required:

These reagents must be purchased separately from the suppliers given in the protocols or in the notes on buffer formulations.

• Nanogold®-Streptavidin - 1 x 500 microliters
• Post-Digestion Buffer - 1 x 25 mL
• Pre-Hybridization Buffer - 1 x 25 m
• 20X Post Hybridization Wash Buffer - 2 x 100 mL
• 10X PBS - 1 x 100 mL
• 10% Blocker - 1 x 5 mL
• Lugol's Iodine - 1 x 25 mL
• Enhancer Reagent - 4 Vials
• Gelatin (45%) - 1 x 1.5 mL
• Development Buffer - 4 Bottles

• Reagent Grade Alcohols
• 2.5% Sodium Thiosulfate
• Ultrapure Water
• Proteinase K (Boehringer Mannheim Cat. # 1373196)
• Biotinylated DNA Probe
• Counterstain
• Mounting Media

• Coplin Jars or equivalents
• Standard Laboratory Pipettors
• Slide Warmers and/or incubators (37°C and 50°C capable)
• Heating Block (92°C-95°C capable)

• Hydrogen Peroxide
• Methanol
• Tween-20
• DMSO
• FITC or Biotin Haptenated Probe
• Streptavidin-Biotin-Peroxidase Complex TSA Kit (NEN Life Sciences, Boston, MA: 1-800-551-2121), which includes:
  • Blocking Powder
  • Biotinylated Tyramide

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Buffer Preparation

Post-Hybridization Wash Buffer:

Four concentrations will be required - 4X, 2X 0.1X, and 0.05X.

For 4X Post-Hybridization Wash Buffer:

Add the contents of one bottle of 20X Post-Hybridization Wash Buffer (100 mL) to 400 mL of ultrapure water to yield 500 mL of 4X Post-Hybridization Wash Buffer.

For 2X Post-Hybridization Wash Buffer:

Add 100 mL of 4X Post-Hybridization Wash Buffer to 100 mL of ultrapure water to yield 200 mL of 2X Post-Hybridization Wash Buffer.

For 0.1X Post-Hybridization Wash Buffer:

Add 10 mL of 2X Post-Hybridization Wash Buffer to 190 mL of ultrapure water to yield 200 mL of 0.1X Post-Hybridization Wash Buffer.

For 0.05X Post-Hybridization Wash Buffer:

Add 100 mL of 0.1X Post-Hybridization Wash Buffer to 100 mL of ultrapure water to yield 200 mL of 0.05X Post-Hybridization Wash Buffer.

1X PBS:

Prepare 1X PBS by adding the contents of the 100 mL bottle of 10X PBS to 900 mL of ultrapure water.

Preparation of TBST (10X concentrated):

• Tris 3.029 g
• Sodium chloride 17.532 g
• Tween-20 5 ml

Make up to 500 mL with double distilled water and adjust pH to 7.6.

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DNA In Situ Hybridization Procedure (without "CARD" System)

Follow instructions provided with probe (or probe preparation kit). Alternatively, use the method described below. Washes are to be done in Coplin jars or equivalents. For all reagent incubations, apply enough reagent so that the specimen is never allowed to dry out during incubation (usually between 0.5 mL and 1.0 mL depending upon specimen size). Between application of different reagents, carefully dry the area surrounding the specimen without disturbing it.

Based on silver/gold-enhanced Nanogold® (Nanoprobes, Inc.). Modified after Hacker et al., Cell Vision, 4, 54-67 (1997), and Zehbe et al., Am. J. Pathol., 150 1553-61 (1997).

1. Formalin fixed/paraffin embedded tissue sections must be deparaffinized and rehydrated by washing as follows:
   1. Fresh Xylenes - 2 x 15 minutes
   2. 100% Alcohol - 2 x 2 minutes
   3. 70% Alcohol - 1 x 2 minutes
   4. 50% Alcohol - 1 x 2 minutes
   5. Ultrapure Water - 2 x 3 minutes
   6. Soak in phosphate-buffered saline (PBS, 20mM, pH 7.6) for 3 min.

2. Shake off the excess and apply Proteinase K dissolved to 0.05% in 1X PBS and incubate at 37°C for about 8 min. The duration is critical and has to be tested very carefully, depending on tissue, fixation and other factors.

3. Rinse in 2 changes of PBS, 3 min.

4. Permeabilize with 0.3% Triton X-100 in PBS for 15 min.

5. Wash in PBS for 2 min.

6. Dehydrate the slide as follows and allow to air-dry:
   1. Ultrapure Water - 2 Minutes
   2. 50% Alcohol - 1 Minute
   3. 70% Alcohol - 1 Minute
   4. 100% Alcohol - 1 Minute

7. Prehybridize with 1:1 mixture of deionized formamide and 20% dextran sulfate in 2X SSC at 50°C for 5 min.

8. Carefully shake off excess prehybridization block.

9. Add one drop of biotinylated DNA probe on the section and cover with a small coverslip. Avoid air bubbles.

10. Heat sections on heating block at 92-94 C for 8-10 min to denature DNA.

11. Incubate in a moist chamber at 37°C overnight (or for at least 2 hours).

12. Post-hybridization washes (5 min each): Wash the slide in Coplin Jars or equivalents as follows (coverslips will fall off during wash):
    1. 2 changes of 4X SSC (1st wash to remove coverslips).
    2. 2X SSC
    3. 0.1X SSC
    4. 0.05X SSC
    5. Distilled Water - 2 minutes

    Detection Procedure

13. Apply Lugol's Iodine and incubate for 5 minutes at room temperature.

14. Wash in tap water and then distilled water.

15. Put into 2.5% sodium thiosulfate for a few seconds until sections are colorless. Then wash in tap water for 5 min and distilled water for 2 min.

16. Immerse in PBS containing 0.1% fish gelatin (45% concentrate - Cat. No. G-7765, Sigma-Aldrich, Steinheim, Germany) and 0.1% Tween-20 for 5 min.

17. Nanogold®-Streptavidin Detection:

    Determine total volume of reagent required for the detection step (usually 0.5 mL - 1.0 mL per specimen). Incubate sections with streptavidin-Nanogold (Nanoprobes, Stony Brook, NY, USA) diluted 1:200 to 1:500 in PBS containing 1% BSA at room temperature for 60 min.

18. Wash specimen as follows:
    1. 3 changes of PBS containing 0.1% fish gelatin and 0.1% Tween-20 for 5 min each.
    2. Repeatedly wash in distilled water for at least 10 min altogether, the last 2 rinses in ultrapure water (EM-grade).

19. Silver Development:

    IMPORTANT: Be sure specimens are well rinsed in water before going on to the silver development step. Also be sure that the containers used for the silver development step are clean and free of all salts and metals.

    To 39 mL of ultrapure water add one vial of Enhancer Reagent (rinse the vial with 1 mL of water and add to the solution) and the intiator reagent, and gently agitate to dissolve contents. To 35 mL of ultrapure water add one bottle of Development Buffer (rinse the bottle with 5 mL of water and add to the solution) and gently agitate to dissolve contents. Add the dissolved Enhancer Reagent to the dissolved Development Buffer in a Coplin jar and then gently agitate to mix. Development begins when all components are mixed. Therefore, use immediately after mixing.

20. Soak the slide in the Coplin jar containing the mixed development reagents until optimal staining is achieved. Progress should be monitored periodically under a light microscope. Seven to ten minutes is an average development time.

21. When optimal staining is achieved stop development by immersing slide in ultrapure water.

22. Rinse slide in water for 5 minutes. Counterstain with 1% eosin or nuclear fast red then soak slides as follows:
    1. Ultrapure water - 2 minutes
    2. 50% alcohol- 1 minute
    3. 70% alcohol - 1 minute
    4. 90% alcohol - 1 minute
    5. 100% alcohol - 1 minute

23. Mount slide using Permount or DPX® (BDH Chemicals) or other suitable mounting reagent and view under a light microscope (Note: Do NOT use Eukitt).

Protocol for Nanogold®-Silver In Situ Hybridization Kit in Conjunction with "CARD" System

Please Note: The TSA Kits have shown some batch variation. Therefore the following procedures using this system are to be used as a guideline only.

DNA In Situ Hybridization Procedure (with "CARD" System)

Follow instructions provided with probe (or probe preparation kit). Alternatively, use the method described below. Washes are to be done in Coplin jars or equivalents. For all reagent incubations, apply enough reagent so the specimen is never allowed to dry out during incubation (usually between 0.5 mL and 1.0 mL depending upon specimen size). Between application of different reagents carefully dry the area surrounding the specimen without disturbing it.

Procedure using NEN Biotinylated Tyramides

Published by Hacker et al., Cell Vision, 4, 54-67 (1997), and Zehbe et al., Am. J. Pathol., 150, 1553-1561 (1997). Follow-up papers: Zehbe I. et al., Am. J. Pathol., 151, 1173 (1997); Hacker G.W., Eur. J. Histochem., 42, 111-120 (1998); Tbakhi A. et al., Am. J. Pathol., 152, 35-41 (1998); Tubbs R.R. et al., Modern Pathology, 11, 594 (1998); Cheung A. et al., Modern Pathology, 12, 689-696 (1999).

1. Formalin fixed/paraffin embedded tissue sections must be deparaffinized and dehydrated by washing as follows:
   1. Fresh Xylenes - 2 x 15 minutes
   2. 100% Alcohol - 2 x 5 minutes

2. Treat with 3% H₂O₂ in methanol at room temperature for 30 min.

3. Rinse in double distilled (ultrapure) water for 10 s and then in PBS for 3 min.

4. Incubate sections with 0.1 mg/mL proteinase K (Boehringer Mannheim, FRG, code # 1 373 196) in PBS at 37°C for about 8 min (optimal duration should be tested). This treatment may partly destroy tissue morphology. However, it is necessary to open-up binding sites for the probe to reach full sensitivity. Combination with microwave treatment may be feasible.

5. Wash in 2 changes of PBS, 3 min each, then ultrapure water for 10 s.

6. Dehydrate the slide as follows:
   1. 50% Alcohol - 5 minutes
   2. 70% Alcohol - 5 minutes
   3. 100% Alcohol - 2 x 5 minutes

7. Allow slide to air-dry. Prehybridize with 1:1 mixture of deionized formamide and 20% dextran sulfate in 2X SSC at 50°C for 5 min.

8. Carefully shake off the excess prehybridization block.

9. Add one drop of biotinylated DNA probe on the section and cover with a small coverslip. Avoid air bubbles.

10. Heat sections on heating block at 92-94°C for 8-10 min to denature DNA.

11. Incubate in a moist chamber at 37°C overnight (or for at least 2 hours).

12. Wash slide in Coplin Jars or equivalents as follows (coverslips will fall off) (5 mins each):
    1. 2 changes of 2X SSC (1st wash to remove coverslips)
    2. 0.5X SSC
    3. 0.2X SSC
    4. Distilled water.

    Detection Procedure

13. Put slides into Lugol's iodine solution (Merck) for 5 min.

14. Wash in tap water and then double distilled water.

15. Put into 2.5% sodium thiosulfate for a few seconds until sections are colorless. Then wash in distilled water for 2 min.

16. Drain off section, wipe area around section dry, and surround it with a PAP-pen (Dako-Pen, Cat. No. S-2002, Dako, Glostrup, DK).

17. Incubate with blocking solution at 37°C for 30 min. Blocking solution is 4X SSC containing 5% casein sodium salt (Cat. No. C-8654, Sigma) or 0.5% blocking powder (from Renaissance TSA-indirect ISH kit, Cat. No. NEL730, NEN Life Science Products, Boston, MA, USA).

18. Brief wash in 4X SSC containing 0.05% Tween-20 (Cat. No. 1332 465, Boehringer Mannheim), 2 min.

19. Incubate with streptavidin-biotin-peroxidase complex (e.g. from Dako duet kit, Dako) at room temperature for 30 min. The complex is dissolved in the above blocking solution at a concentration of 1:200.

20. Wash as follows:
    1. 3 changes of 4X SSC containing 0.05% Tween-20 for 2 min each.
    2. 2 changes of PBS for 2 min each.

21. Incubate the sections with biotinylated tyramide (BT) at room temperature for exactly 10 (with BT from Renaissance TSA-indirect ISH kit, NEN) or 15 min (with BT from GenPoint kit, Cat. No. K0620, Dako). The BT reagent in the Dako kit is ready-to-use. For the NEN-kit, a stock solution of BT is prepared by adding 100 mL ethanol to the lyophilized reagent and is diluted 1/50 to 1/100 with the supplied amplification diluent mixed with distilled water at 1:1, as described in the kit. According to the guidelines supplied, the working solution should contain 1 or 0.5 mg of BT per mL diluent, consisting of 0.2 mol/L Tris-HCl, 10 mmol/L imidazole, pH 8.8 and 0.01% H₂O₂.

22. Wash in 4 changes of PBS containing 0.05% Tween-20 and 20% DMSO (dimethyl sulfoxide) at room temperature for 3 min each.

23. Nanogold®-Streptavidin Detection:

    Immerse in PBS-gelatin (PBS containing 0.1% fish-gelatin) for 5 min. A 45% fish-gelatin concentrate is available from Sigma-Aldrich, code # G-7765.

24. Determine total volume of reagent required for the detection step (usually 0.5 mL - 1.0 mL per specimen). Incubate the sections with streptavidin-Nanogold (Nanoprobes) diluted 1:750 in PBS containing 1% BSA at room temperature for 60 min.

25. Wash in 3 changes of PBS-gelatin for 5 min each.

26. Repeatedly wash in ultrapure water (EM-grade).

27. Perform silver acetate autometallography (AMG) or GoldEnhance development (Nanoprobes) according to the instructions of the manufacturer.

    NOTE: For all applications where Nanogold® is used instead of colloidal gold, repeated washes in distilled water are usually sufficient to stop autometallographic development when using either silver acetate autometallography or GoldEnhance. However, if development is found to continue even after repeated water washes (several changes), and background staining becomes excessive, slides may be dipped briefly into freshly prepared 2.5% sodium thiosulfate. If this is used, care should be taken not to reduce the specific signals.

28. Silver Development:

    IMPORTANT: Be sure specimens are well rinsed in water before going on to the silver development step. Also be sure that the containers used for the silver development step are clean and free of all salts and metals.

    To 39 mL of ultrapure water add one vial of Enhancer Reagent (rinse the vial with 1 mL of water and add to the solution) and the intiator reagent, and gently agitate to dissolve contents. To 35 mL of ultrapure water add one bottle of Development Buffer (rinse the bottle with 5 mL of water and add to the solution) and gently agitate to dissolve contents. Add the dissolved Enhancer Reagent to the dissolved Development Buffer in a Coplin jar and then gently agitate to mix. Development begins when all components are mixed. Therefore, use immediately after mixing.

29. Soak the slide in the Coplin jar containing the mixed development reagents until optimal staining is achieved. Progress should be monitored periodically under a light microscope. Seven to ten minutes is an average development time.

30. When optimal staining is achieved stop development by immersing slide in ultrapure water for 3 minutes. Do Not use sodium thiosulfate or a photographic fixer for the "CARD" system.

31. Rinse slide in water for 5 minutes. Counterstain with 1% eosin or nuclear fast red then soak slides as follows:
    1. Ultrapure water - 2 minutes
    2. 50% alcohol- 1 minute
    3. 70% alcohol - 1 minute
    4. 90% alcohol - 1 minute
    5. 100% alcohol - 1 minute

32. Mount slide using Permount or DPX® (BDH Chemicals) or other suitable mounting reagent and view under a light microscope (Note: Do NOT use Eukitt).

Procedure using Dako Biotinylated Tyramides

1. Deparaffinize sections from formaldehyde-fixed tissue in fresh xylene (2 times 15 min each).

2. Rinse in absolute ethanol (2 times 5 min each), then 95% ethanol (2 times 5 min each), followed by 2 changes of double distilled water.

3. Immerse the slides in Target Retrieval solution (DAKO Cat. # S1700) at 95°C for 40 min, then let the slides cool in the same solution for 20 min.

4. Rinse the slides in several changes of double distilled water, then incubate them with proteinase K (DAKO Cat. #S3004) diluted 1:5000 in 50 mM Tris-HCl buffer (pH 7.6) for 5 min at room temperature. Alternatively, steps 3-4 may be replaced by pretreatment with 0.1 mg/mL proteinase K (Boehringer Mannheim, FRG, code # 1 373 196) in 50 mM Tris-HCl buffer (pH 7.6) at 37°C for about 8 min (optimal duration should be tested).

5. Wash slides in double distilled water (3 changes, 5 min each).

6. Treat with 3% H₂O₂ in methanol at room temperature for 30 min.

7. Wash slides in double distilled water for 10 min.

8. Put slides into Lugol's iodine solution (Merck) for 5 min, then wash in double distilled water.

9. Put into 2.5% sodium thiosulfate for a few seconds until sections are colorless, then wash in double distilled water (2 times, 5 min each).

10. Allow slides to air-dry.

11. Add one drop of biotinylated DNA probe on the section and cover with a small glass coverslip. Avoid air bubbles.

12. Heat sections on heating block at 92-94°C for 5 min to denature DNA.

13. Incubate in a moist chamber at 37°C overnight (or for at least 1 hour).

14. Remove the coverslip by soaking slides in a TBST (Tris buffered saline containing Tween 20) bath for 5 min.

15. Incubate slides in "Stringent Wash" (provided in the GenPoint Kit) for 20 min at 55°C.

16. Drain off section, wipe area around section dry, and surround it with a PAP-pen (Dako-Pen, Cat. No. S-2002, Dako, Glostrup, DK).

17. Immerse slides in TBST for 5 min.

18. Apply primary streptavidin-HRP, diluted 1:800 in the diluent (from GenPoint Kit) to sections and incubate in a moist chamber for 15 min at room temperature.

19. Wash in 3 changes of TBST, 5 min each.

20. Apply ready-to-use biotinyl-tyramide solution (from GenPoint Kit) and incubate in a moist chamber for 15 min at room temperature.

21. Wash in 3 changes of TBST-gelatin (TBST containing 0.1% fish gelatin (Sigma), pH 7.6), for 5 min each.

22. Incubate the sections with streptavidin-Nanogold® (Nanoprobes) diluted 1:250 in PBS containing 1% BSA at room temperature for 60 min.

23. Wash in 3 changes of TBST-gelatin for 5 min each.

24. Repeatedly wash in ultrapure water (EM-grade).

25. Perform silver acetate autometallography (AMG) or GoldEnhance development (Nanoprobes) according to the instructions of the manufacturer.

    NOTE: For all applications where Nanogold® is used instead of colloidal gold, repeated washes in distilled water are usually sufficient to stop autometallographic development when using either silver acetate autometallography or GoldEnhance. However, if development is found to continue even after repeated water washes (several changes), and background staining becomes excessive, slides may be dipped briefly into freshly prepared 2.5% sodium thiosulfate. If this is used, care should be taken not to reduce the specific signals.

26. Silver Development:

    IMPORTANT: Be sure specimens are well rinsed in water before going on to the silver development step. Also be sure that the containers used for the silver development step are clean and free of all salts and metals.

    To 39 mL of ultrapure water add one vial of Enhancer Reagent (rinse the vial with 1 mL of water and add to the solution) and the intiator reagent, and gently agitate to dissolve contents. To 35 mL of ultrapure water add one bottle of Development Buffer (rinse the bottle with 5 mL of water and add to the solution) and gently agitate to dissolve contents. Add the dissolved Enhancer Reagent to the dissolved Development Buffer in a Coplin jar and then gently agitate to mix. Development begins when all components are mixed. Therefore, use immediately after mixing.

27. Soak the slide in the Coplin jar containing the mixed development reagents until optimal staining is achieved. Progress should be monitored periodically under a light microscope. Seven to ten minutes is an average development time.

28. When optimal staining is achieved stop development by immersing slide in ultrapure water for 3 minutes.

    NOTE: For all applications where Nanogold® is used instead of colloidal gold, repeated washes in distilled water are usually sufficient to stop autometallographic development when using either silver acetate autometallography or GoldEnhance. However, if development is found to continue even after repeated water washes (several changes), and background staining becomes excessive, slides may be dipped briefly into freshly prepared 2.5% sodium thiosulfate. If this is used, care should be taken not to reduce the specific signals.

29. Rinse slide in water for 5 minutes. Counterstain with 1% eosin or nuclear fast red then soak slides as follows:
    1. Ultrapure water - 2 minutes
    2. 50% alcohol- 1 minute
    3. 70% alcohol - 1 minute
    4. 90% alcohol - 1 minute
    5. 100% alcohol - 1 minute

30. Mount slide using Permount or DPX® (BDH Chemicals) or other suitable mounting reagent and view under a light microscope (Note: Do NOT use Eukitt).

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RNA In Situ Hybridization Procedure (with "CARD" System)

Follow instructions provided with probe (or probe preparation kit). Alternatively use the method described below. Washes are to be done in Coplin jars or equivalents. For all reagent incubations, apply enough reagent so the specimen is never allowed to dry out during incubation (usually between 0.5 mL and 1.0 mL depending upon specimen size). Between application of different reagents carefully dry the area surrounding the specimen without disturbing it.

Published in Hacker et al., Cell Vision, 4 54-67 (1997). Follow-up papers: Hacker G.W., Eur. J. Histochem., 42, 111-120 (1998); Tbakhi A. et al., Am. J. Pathol., 152, 35-41 (1998); Tubbs R.R. et al., Modern Pathology, 11, 594 (1998); Hacker G.W., Zehbe I., Tubbs R.R.: "Supersensitive in situ DNA and RNA localization." In: In situ hybridization: Principles and Practice. Second Edition (Edited by Julia M. Polak and James OD. McGee), Oxford University Press, Oxford, UK, pp. 179-206, 1998.

1. Formalin fixed/paraffin embedded tissue sections must be deparaffinized and dehydrated by washing as follows:
   1. Fresh Xylenes - 2 x 15 minutes
   2. 100% Alcohol - 2 x 5 minutes

2. Treat with 3% H₂O₂ in methanol at room temperature for 30 min.

3. Rinse in double distilled (ultrapure) water for 10 s and then in PBS for 3 min.

4. Incubate sections with 0.1 mg/mL proteinase K in PBS at 37°C for about 8 min. Optimal concentration and duration is critical and should be tested carefully. Supplier: Boehringer Mannheim, Germany, code # 1 373 196). This treatment may partly destroy tissue morphology. However, it is necessary to open-up binding sites for the probe to reach full sensitivity. Combination with microwave treatment may be feasible.

5. Wash in 2 changes of PBS, 3 min each, then ultrapure water for 10 s.

6. Dehydrate the slide as follows:
   1. 50% Alcohol - 5 minutes
   2. 70% Alcohol - 5 minutes
   3. 100% Alcohol - 5 minutes

7. Allow slide to air-dry. Prehybridize with 1:1 mixture of deionized formamide and 20% dextran sulfate in 2X SSC at 50°C for 5 min.

8. Carefully shake off the excess prehybridization block.

9. Add one drop of FITC haptenated ribonucleotide or antisense oligonucleotide probe on the section and cover with a small coverslip. Avoid air bubbles.

10. Hybridize in a moist chamber at 37°C overnight (or for at least 2 hours).

11. Wash slide in Coplin Jars or equivalents as follows (coverslips will fall off):
    1. 2 changes of 2X SSC (1st wash to remove coverslips), 5 minutes each
    2. 0.5X SSC - 5 minutes
    3. 0.2X SSC - 5 minutes
    4. Distilled water - 5 minutes.
    5. 1X PBS - 3 minutes

    Detection Procedure

12. Drain off section, wipe area around section dry, and surround it with a PAP-pen (Dako-Pen, Cat. No. S-2002, Dako Glostrup, DK).

13. Incubate with mouse monoclonal anti-FITC antibody (Boehringer Mannheim, code # BA-9200) diluted in PBS at a working dilution of 1:1000 at RT for 30 min.

14. Wash in PBS twice for 3 min each.

15. Incubate with biotinylated goat anti-mouse IgG antibody (Vector, Burlingame, USA) diluted in PBS at a working dilution of 1:200 at RT for 30 min.

16. Wash in PBS twice for 3 min each.

17. Apply Lugol's Iodine and incubate for 5 minutes at room temperature. Wash slide in ultrapure water. Apply 2.5% sodium thiosulfate to the slide until specimen becomes colorless (usually only a few seconds). Wash in ultrapure water 3 times for 3 minutes per wash.

18. Wash in tap water and then double distilled water.

19. Incubate with blocking solution at 37°C for 30 min. Blocking solution is 4X SSC containing 5% casein sodium salt (Cat. No. C-8654, Sigma-Aldrich, Steinheim, Germany) or 0.5% blocking powder (from Renaissance TSA-indirect ISH kit, Cat. No. NEL730, NEN Life Science Products, Boston, MA, USA).

20. Brief wash in 4X SSC containing 0.05% Tween-20 (Cat. No. 1332 465, Boehringer Mannheim), 2 min.

21. Incubate with streptavidin-biotin-peroxidase complex (e.g. from Dako duet kit, Dako) at RT for 30 min. The complex is dissolved in the above blocking solution at a concentration of 1:200.
    1. 3 changes of 4X SSC containing 0.05% Tween-20 for 2 min each
    2. 2 changes of PBS for 2 min each.

22. Incubate the sections with biotinylated tyramide (BT) at room temperature for exactly 10 (with BT from Renaissance TSA-indirect ISH kit, NEN) or 15 min (with BT from GenPoint kit, Cat. No. K0620, Dako). For the NEN-kit, a stock solution of BT is prepared by adding 100 mL ethanol to the lyophilized reagent and is diluted 1/50 to 1/100 with the supplied amplification diluent mixed with distilled water at 1:1, as described in the kit. According to the guidelines supplied, the working solution should contain 1 or 0.5 mg of BT per mL diluent, consisting of 0.2 mol/L Tris-HCl, 10 mmol/L imidazole, pH 8.8 and 0.01% H₂O₂.

23. Wash in 4 changes of PBS containing 0.05% Tween-20 and 20% DMSO (dimethyl sulfoxide) at room temperature for 3 min each.

24. Nanogold®-Streptavidin Detection:

25. Immerse in PBS-gelatin (PBS containing 0.1% fish-gelatin) for 5 min. A 45% fish-gelatin concentrate is available from Sigma-Aldrich, code # G-7765.

26. Determine total volume of reagent required for the detection step (usually 0.5 mL - 1.0 mL per specimen). Use this volume of 1X PBS and into this dilute the 10% Blocker to 1%. Into this dilute the Nanogold®-Streptavidin 1:750 (e.g., for 15 mL add 20 microliters of Nanogold®-Streptavidin). Incubate the sections with the 1:750 diluted streptavidin-Nanogold® (Nanoprobes) in PBS containing 1% BSA at room temperature for 60 min.

27. Wash in PBS-gelatin for 5 min (3 times 5 min each).

28. Repeatedly wash in ultrapure water (EM-grade).

29. Silver Development:

    IMPORTANT: Be sure specimens are well rinsed in water before going on to the silver development step. Also be sure that the containers used for the silver development step are clean and free of all salts and metals.

    To 39 mL of ultrapure water add one vial of Enhancer Reagent (rinse the vial with 1 mL of water and add to the solution) and the intiator reagent, and gently agitate to dissolve contents. To 35 mL of ultrapure water add one bottle of Development Buffer (rinse the bottle with 5 mL of water and add to the solution) and gently agitate to dissolve contents. Add the dissolved Enhancer Reagent to the dissolved Development Buffer in a Coplin jar and then gently agitate to mix. Development begins when all components are mixed. Therefore, use immediately after mixing.

30. Soak the slide in the Coplin jar containing the mixed development reagents until optimal staining is achieved. Progress should be monitored periodically under a light microscope. Seven to ten minutes is an average development time.

31. When optimal staining is achieved stop development by immersing slide in ultrapure water for 3 minutes.

    NOTE: For all applications where Nanogold® is used instead of colloidal gold, repeated washes in distilled water are usually sufficient to stop autometallographic development when using either silver acetate autometallography or GoldEnhance. However, if development is found to continue even after repeated water washes (several changes), and background staining becomes excessive, slides may be dipped briefly into freshly prepared 2.5% sodium thiosulfate. If this is used, care should be taken not to reduce the specific signals.

32. Rinse slide in water for 5 minutes. Counterstain with 1% eosin or nuclear fast red then soak slides as follows:
    1. Ultrapure water - 2 minutes
    2. 50% alcohol- 1 minute
    3. 70% alcohol - 1 minute
    4. 90% alcohol - 1 minute
    5. 100% alcohol - 1 minute

33. Mount slide using DPX (BDH Chemicals) or other suitable mounting reagent and view under a light microscope.

NOTES

Reagent and Buffer Formulations

Pre-Digestion Buffer: 0.1% Triton X-100 in PBS
Pre-Hybridization Buffer: 50% deionized formamide / 10% dextran sulfate in 2X SSC
20X Post-Hybridization Wash Buffer: 20X SSC (175.32g NaCl / 88.23g NaCitrate to 1 L with water)
Lugol's Iodine: Sigma Lugol's Iodine (Cat. No. L-6146)
Gelatin (45%): Sigma Cold Water Fish Gelatin (Cat. No. G-7765)
Nanogold®-Streptavidin: Nanoprobes (Cat. No. 2016)
10% Blocker: 10% BSA in water (filtered)
10X PBS: 0.2M NaPhosphate / 1.5M NaCl pH 7.6
Enhancer Reagent - 80 mg Silver Acetate
Development Buffer: 1.1g Trisodium Citrate / 1.4g Citric Acid
Initiator Reagent: 200 mg Hydroquinone

References

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2. Hacker, G. W., Hauser-Kronberger, C., Zehbe, I., Su, H., Schiechl, A., Dietze, O., and Tubbs, R.: In Situ localization of DNA and RNA sequences: Super-sensitive In Situ hybridization using Streptavidin-Nanogold-Silver Staining: Minireview, Protocols and Possible Applications. Cell Vision, 4, 54-65 (1997).

3. Hacker, G. W.; Hauser-Kronberger, C.; Zehbe, I.; Su, H., and Tubbs, R.: New advances in super-sensitive DNA-, RNA- and antigen detection: Combination of labeled tyramides with Nanogold-silver staining (NGSS). Proc. 56^th Ann. Mtg., Micros. Soc. Amer.; Bailey, G. W.; Alexander, K. B.; Jerome, W. G.; Bond, M. G., and McCarthy, J. J., Eds.; Springer, New York, NY, 1998, 996-997.

4. Hacker, G. W., Zehbe, I., Hainfeld, J., Graf, A.-H., Hauser-Kronberger, C., Schiechl, A., Su, H., and Dietze, O.: High performance Nanogold®-in situ hybridization and its use in the detection of hybridized and PCR-amplified microscopical preparations; In Proc 54th Ann. Mtg. Micros. Soc. Amer., G. W. Bailey, J. M. Corbett, R. V. W. Dimlich, J. R. Michael and N. J., Zaluzec (Eds.). San Francisco Press, San Francisco, CA, pp. 896-897 (1996).

5. Hacker, G. W.; Zehbe, I.; Hainfeld, J.; Sällström, J.; Hauser-Kronberger, C.; Graf, A.-H.; Su, H.; Dietze, O., and Bagasra, O; High-Performance Nanogold® In Situ Hybridization and In Situ PCR. Cell Vision, 3, 209 (1996).

   Download this paper as a PDF file from the Microscopy Society of America.

6. Hauser-Kronberger, C.: Highly sensitive DNA, RNA and antigen detection methods. Streptavidin-Nanogold-silver staining. Cell Vision, 5, 83 (1998).

7. Punnonen, E.-L., Fages, C., Wartiovaara, J., and Rauvala, H.: Ultrastructural Localization of beta-Actin and Amphoterin mRNA in Cultured Cells: Application of tyramide signal amplification and comparison of detection methods; J. Histochem. Cytochem., 47, 99 (1999).
   • Abstract (from JHC Online).

8. Schöfer, C.; Weipoltshammer, K.; Hauser-Kronberger, C., and Wachtler, F.: High-resolution detection of nucleic acids at the electron microscope level - Review of in situ hybridization technology, the use of gold, and Catalyzed Reporter Deposition (CARD). Cell Vision, 4, 443-454 (1997).

9. Tbakhi, A.; Totos, G.; Hauser-Kronberger, C.; Pettay, J.; Baunoch, D.; Hacker, G. W., and Tubbs, R. R.: Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma. Am. J. Pathol., 152, 35-41 (1998).

10. Totos, G.; Tbakhi, A.; Hauser-Kronberger, C., and Tubbs, R. R.: Catalyzed Reporter Deposition: A new era in molecular and immunomorphology - Nanogold-silver staining and colorimetric detection and protocols. Cell Vision, 4, 433-442 (1997).

11. Zehbe, I., Hacker, G.W., Su, H., Hauser-Kronberger, C., Hainfeld, J.F., and Tubbs, R.: Sensitive in situ hybridization with catalyzed reporter deposition, streptavidin-Nanogold, and silver acetate autometallography. Detection of single-copy human papillomavirus. Am. J. Pathol. 150, 1553-1561 (1997).