Materials

1. Clean glass slides (see Note 1).

2. Slide frames or self-sealing solution.

3. Coplin jars and glass staining dishes.

4. 2% Paraformaldehyde: add 12 g of paraformaldehyde (Merck ultra pure Art no. 4005) to 600 mL of 1X phosphate-buffered saline (PBS) and heat at 65°C for

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed. Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

10 min. When the solution starts to clear, add four drops of 10 N NaOH and stir. Adjust to neutral pH and cool to room temperature. Filter on Whatman's no. 1.

5. 10X PBS stock solution, pH 7.2 to 7.4: dissolve 20.5 g of NaH2PO4 • H2O and 179.9 g of Na2HPO • 7H2O (or 95.5 g Na2 HPO4) in approx 4 L of double-distilled water, then adjust to the required pH (7.27.4). Add 701.3 g of NaCl and make up to a total volume of 8 L.

6. 1X PBS: dilute the 10X PBS stock at a 1:10 ratio (i.e., 100 mL of 10X PBS and 900 mL of water for 1 L). Final concentration of buffer should be 0.01 M phosphate and 0.15 M NaCl.

7. 0.3% Hydrogen peroxide (H2O2) in PBS: dilute stock to 30% hydrogen peroxide (H2O2) at a 1:100 ratio in 1X PBS for a final concentration of 0.3% H2O2.

8. Proteinase K: dissolve powder from Sigma in water to obtain 1 mg/mL concentration. Aliquot and store at -20°C. Prepare a working solution (6 |g/mL by diluting 1 mL of stock (1 mg/mL) into 150 mL of 1X PBS.

9. 20X standard saline citrate (SSC): dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 mL of water. Adjust the pH to 7.0 with a few drops of 10 NNaOH. Adjust the volume to 1 L with water. Sterilize by autoclaving.

10. 2X SSC: dilute 100 mL of 20X SSC in 900 mL of water.

11. RPMI Medium 1640: per 100 mL, supplement with 15 mL of fetal bovine serum, 1.5 of HEPES buffer, and 0.1 mL of gentamycin (0.1 heparin is optional).

12. Velban: reconstitute vial with 10 mL of sterile H2O. From this solution, dilute 0.1 mL into 50 mL distilled H2O. Store in the refrigerator.

13. EGTA hypotonic solution: dissolve 0.2 g of EGTA powder, 3.0 g of KCl, and 4.8 g of HEPES buffer into 1000 mL of distilled H2O. Adjust pH to 7.4. Store in refrigerator and, prior to use, prewarm to 37°C.

14. Phytohemagglutinin (PHA-C): reconstitute with 5 mL of sterile H2O. Aliquot into five 1-mL insulin syringes. Freeze four for later use and leave one in refrigerator.

15. Fixative solution: one part glacial acetic acid in three parts methanol; store at -20°C.

16. Streptavidin peroxidase: dissolve powder from Sigma in PBS to make a stock of 1 mg/mL. Just before use, dilute stock solution in sterile PBS at a 1:30 ratio.

17. Color solution: dissolve one tablet of 3-amino-9-ethyl-carbazole (AEC; Sigma) in 2.5 mL of N,N,dimethyl formamide. Store at 4°C in the dark. To prepare working solution, mix the following:

b. AEC solution 250 |L

Make fresh before each use, keeping solution in the dark.

18. 50 mM acetate buffer pH 5.0: add 74 mL of 0.2 Nacetic acid (11.55 mL glacial acid/L and 176 mL of 0.2 M sodium acetate (27.2 g sodium acetate trihydrate in 1 L) to 1 L of deionized water and mix.

19. In situ hybridization buffer (for 5 mL): 2.5 mL formamide, 500 |L of salmon sperm DNA (ssDNA; 10 mg/mL), 500 |L of 20X SSC, 1 mL of 50X Denhardt's solution, 50 ||L of 10% sodium dodecyl sulfate (SDS), and 450 ||L of water. Heat to denature ssDNA at 94°C for 10 min before adding to the solution.

20. P20 Micropipet.

21. Xylene solution (EM grade, benzene free).

22. 2% AES solution: 5 mL of 3-aminopropyltriethoxysilane (AES: Sigma A-3648), and 250 mL of acetone.

23. Ethanol series: 100%, 90%, 80%, 70%, and 50% (EM grade).

24. Tissue-Tek OCT (an embedding media for frozen tissue specimens made by Miles Laboratory of Elkhart, IN).

25. RNase-free, DNase solution: 40 mMTris-HCl, pH 7.4, 6 mMMgCl2, 2 mMCaCl2, and 1 U/|L final volume of DNase (use RNase-free DNase, such as 10 U/|L RQ1 DNase, cat no. 776785 from Boehringer or any other reliable source).

26. 10X Reverse transcriptase (RT) reaction buffer: 100 mM Tris, pH 8.3; 500 mM KCl; 15 mM MgCl2.

27. RT reaction solution (if using Moloney Murine Leukemia Virus [MuLV] RT enzyme, or other equivalent) for a total volume of 20.0 |L:

28.

a.

10X Reaction buffer

2.0 | L

b.

10 mM dATP

2.0 | L

c.

10 mM dCTP

2.0 | L

d.

10 mM dGTP

2.0 | L

e.

10 mM dTTP

2.0 | L

f.

RNasin (40 | /| L)

0.5 | L

g.

20 | M downstream primer

1.0 |L

h.

AMVRT 20 U/| L

0.5 | L

i.

Diethylpyrocarbonate (DEPC) water

8.0 | L

Mixture for one-step RT-amplification (for 100 ||L):

a.

100 |MForward primer

0.5 | L

b.

100 |MReverse primer

0.5 | L

c.

3 mM Nucleotide mix (dNTP) 6.0 |L.

d.

10 mM MnCl2

2.0 | L

e.

25 mM MgCl2

10.0 |L

f.

10X Transcription buffer

2.0 | L

g.

10X Chelating buffer

8.0 | L

h.

1.7 mg/mL Bovine serum albumin (BSA)

10.0 |L

i.

2.5 U/mL rTth enzyme

2.0 | L

j.

DEPC-treated water

59.0 | L

29. 10X Transcription buffer: 100 mM Tris-HCl, pH 8.3; 900 mMKCl.

30. 10X Chelating buffer: 100 mMTris-HCl, pH 8.3; 1 MKCl; 7.5 mMEGTA, 0.5% Tween-20; 50% (v/v) glycerol.

31. Streptavidin-alkaline phosphatase conjugate (40 |g/mL stock).

32. Blocking solution for alkaline phosphatase-based color development: 50 mg/mL BSA (protein) in 100 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.2 mg/mL sodium azide.

33. Conjugate dilution buffer for alkaline phosphatase-based color development: 100 mM Tris-HCl, 150 mMMgCl2, 10 mg/mL BSA, 0.2 mg/mL sodium azide.

34. Buffer A for alkaline phosphatase-based color development: 100 mMTris-HCl, pH 7.5; 150 mM NaCl.

35. Alkaline substrate buffer for alkaline phosphatase-based color development: 100 mM Tris-HCl, pH 9.5, 150 mMNaCl, 50 mMMgCl2.

36. Nitro-blue-tetrazolium (NBT): 75 mg/mL NBT in 70% (v/v) dimethlyformamide, freshly prepared.

37. 4-Bromo-5-chloro-3-indolylphosphate (BCIP): 50 mg/mL in 100% dimethylformamide, freshly prepared.

38. Water-based mounting medium such as CrystalMount™ or GelMount™ or an organic solvent-based medium such as Permount™ (Fisher Scientific).

39. Light microscope (x400).

40. Ultraviolet (UV) microscope with appropriate filter range. 3. Methods

3.1. AES Silanization: Putting on the Positive Charge

1. Prepare the 2% AES solution just before use.

2. Put solution into a Coplin jar or glass staining dish and dip glass slides in 2% AES for 60 s.

3. Dip slides five times into a different vessel filled with 1000 mL of distilled water.

4. Repeat step 3 three times, changing the water each time.

5. Air-dry in laminar-flow hood from a few hours to overnight and then store slides in a sealed container at room temperature. Try to use slides within 15 d of silanation; 250 mL of AES solution is sufficient to treat 200 glass slides.

3.2. Preparation of Tissue

3.2.1. Cell Suspensions

1. Isolate cells on a Ficoll-Hypaque density gradient (tissue-culture cells or other single-cell suspensions can also be used).

2. Wash cells with 1X PBS twice.

3. Resuspend cells in PBS at 2 x 106 or 5 x 105 cells/mL.

4. Add 10 pL of cell suspension to each well of the slide using a P20 micropipet.

5. Air-dry slide in a laminar-flow hood.

3.2.2. Cells Cultured on Slide

The slides must be sterilized after silanization and before innoculation with the culture.

1. Soak the slides in ethanol for 30 min.

2. Cell adhesion substances (i.e., fibronectin, etc) can be added on the slide surface and air-dried.

3. Place the cells on the slides and culture overnight in a sterile, humidified box (see Note 2).

3.2.3. Paraffin-Fixed Tissue

1. Place tissue sections on properly prepared slide, as described previously (see Note 3).

2. Place tissue section upon the glass surface of the slide.

3. Incubate the slides in an oven at 80°C for 1 h to melt the paraffin.

4. Dip the slides in xylene for 5 min, then in 100%, 90%, 80%, 70%, and 50% etha-nol, and then in H2O for 5 min each.

3.2.4. Freezing the Tissue

Three methods of tissue freezing are described.

3.2.4.1. Tissue Freezing Method A

1. Cut a 1 x 1 cm piece of styrofoam from a sheet approx 3-mm thick (or use a disposable styrofoam coffee cup if a sheet is not available).

2. Cut a slice of tissue about the same size as the styrofoam square but somewhat thicker (up to 1 cm thick).

3. Pour approx 2 mL of Tissue-Tek OCT onto the styrofoam substrate.

4. Lay on the tissue.

5. Pour another 2 mL of OCT on top so that the tissue is covered with OCT.

6. Fashion an immersion tool from wire or a coat hanger so that the styrofoam/ tissue sample can rest on a loop of wire with a wire handle that allows immersion.

7. Slowly lower the apparatus and sample into the liquid nitrogen; the tissue should freeze in less than 30 s.

8. Load the tissue into a cryocassette.

9. Cut or put into a -70°C freezer for storage.

3.2.4.2. Tissue Freezing Method B

1. Place the tissue into a small plastic bag designed for immersion into liquid nitrogen. These bags are common items in pathology laboratories.

2. Immerse the tissue in the liquid nitrogen.

3. Remove and either load into a cryocassette for cutting or put into a -70°C freezer for storage.

3.2.4.3. Tissue Freezing Method C

1. If liquid nitrogen is not available, tissue prepared with styrofoam and OCT (as in method A) can be wrapped in aluminum foil.

2. Place on dry ice for 10 to 15 min before storing in the deep freezer. However, some ice crystallization in the tissue may occur with this method.

3. Under no circumstance attempt to freeze the tissue by merely placing it into a -70°C freezer. This will result in an abundance of ice crystals, and the sections will not be suitable for in situ procedures (see Note 4).

3.2.5. Sectioning the Tissue

1. It is necessary to use as thin a section as possible, that is, as small as 6 pL in thickness.

2. Once the tissue is properly sliced (see Note 5), apply to slide.

3. Dehydrate for 10 min in 100% methanol.

4. Air-dry in a laminar flow hood.

5. Proceed to the heat-treatment step (i.e., Subheading 3.3.1.).

3.2.6. Archival Tissue

1. Remove the cover slip and mounting by soaking in 100% methanol (or carefully in acetone if methanol does not pry the cover slips open).

2. Deparafinnize as described previously in Subheading 3.2.3.

3. Because the tissue is already on a slide, one does not have a choice about the area of the slide covered by the section or what type of surface preparation to use.

4. Instead, just develop the chamber with the Frame Chamber, as shown in Fig. 1.

5. Load the reagents into the wells of the chamber with PCR cocktail and seal them with the plastic sealing cover paper provided.

6. Not all of the archival tissue will be subject to the amplification-hybridization procedure, but usually there is a sufficient area in the well region to get adequate results.

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