Carlos A. Muro-Cacho, M.D., Ph.D.

Department of Pathology, H. Lee Moffitt Cancer Center and Research Institute and University of South Florida College of Medicine, Tampa, Florida, USA

Received 1/10/97 Accepted 7/15/97

10. APPENDICES

Appendix 1 – General information

A typical In situ PCR protocol for the detection of DNA (i.e., of viral origin), in tissue sections, will start with the collection and preparation of the sample. This requires optimal fixation of the tissue, followed by tissue embedding, generally in paraffin. The tissue is then sectioned and placed onto a slide. Standard deparaffination protocols are followed by enzymatic digestion to allow penetration of reagents. The tissue is covered with a PCR solution containing specific primers, DNA polymerase, and a mixture of nucleotides in an appropriate buffer. Amplification of the desired nucleic acid sequences is achieved using a cycling profile determined to be optimal for the particular conditions of the experiment. Detection of the amplified material can be done indirectly, by In situ Hybridization, using a labeled specific probe or, directly, by incorporating a labeled nucleotide during the amplification step. The label generally used is digoxigenin which can be detected by an immunoenzymatic method. A colored product is deposited at the site of the amplified nucleic acid sequences. If the sequence to be detected is mRNA, prior to the In situ PCR, the tissue section may be exposed to DNase, to destroy the endogenous DNA. In a reverse transcription step, mRNA is converted into c-DNA that is further amplified and detected as described.

Appendix 2 - Sample preparation

Cells

1. Wash cells three times x 5 minutes in PBS at 4^o C


2. Resuspend cells in PBS at the desired concentration (i.e., 2 million cells/ml)


3. Add 50 microliters onto a slide and air dry or prepare cytospin


4. Fix in 95 % ethanol, 4 % paraformaldehyde or 10 % neutral buffered formalin x 2-15 hours


5. If slides are to be stored, air dry after dehydration and rehydrate prior to use


6. Prior to proceeding with the protocol, wash in PBS and water

Frozen sections

1. Place sections, 5-10 microns in thickness, onto appropriately coated slides


2. Wash twice with PBS at 4^o C


3. Fix in 4% paraformaldehyde or 10 % neutral buffered formalin x 2-18 hours


4. Dehydrate through ethanol, air dry, and store at 4^o C


5. Rehydrate prior to use

Paraffin-embedded tissue sections

1. Prepare 4-5 microns tissue sections onto coated glass slides


2. Adhere section to slide by heating on a 60^o C hot plate for 12 hours


3. Deparaffinize and rehydrate following standard protocols


4. Incubate slides in 0.02M HCl for 10 minutes


5. Wash slides twice in PBS for 5 minutes each


6. Inmerse slides in 0.01 % (v/v) Triton-X 100 in PBS for 2 minutes


7. Wash slides twice in PBS for 5 minutes each


8. Proceed with proteolytic digestion (appendix 3)

Appendix 3 - Proteolytic digestion

Materials - Proteolytic solutions

Proteinase K: 10 to 20 micrograms/milliliter in 0.1M Tris-HCl pH 7.5, 5mM EDTA x 20-30 minutes at 37^o C(aliquots can be stored at –20^o C). Stop activity with a solution of 20 % acetic acid at 4^o C x 10 seconds or with 0.1 M Glycine in PBS x 5 min.

Trypsin: 0.1 % solution in 0.4 % calcium chloride x 30 minutes at room temperature. Inactivate in 0.1 M Tris at pH 7.4.

Pepsin: 0.4 % in 0.04M hydrochloric acid x 30 minutes at room temperature.

Method

1. Incubate sections in buffer at 37^o C


2. Add proteolytic solution, coverslip and digest


3. Remove coverslip and inactivate enzyme


4. Wash in PBS and water


5. Dehydrate to 100 % ethanol


6. Proceed with protocol or store in 100 % ethanol at 4^o C until use

Materials

DNAse I (Life Technologies, Gaithersburg, MD) 0.1 U/microliter in 20 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, and 100 micrograms/milliliter BSA w/v. Incubate overnight at room temperature, in a humidified chamber. Stop reaction with 20 mM EDTA for 10 min at 65^o C plus three 10 minutes washes in 50 mM Tris-HCl, pH 7.5.

RNAse-free DNAse (Boehringer Mannheim, Indianapolis, IN) 1 U/microliter in a solution containing 40 mM Tris-HCl pH 7.4, 6 mM MgCl₂, and 2 mM CaCl₂. Incubate overnight at 37^o C in a humidified chamber. Rinse slides for 1 min in DEPC-treated water, wash x 1 min in 100 % ethanol and air dried.

Method

1. Incubate sections in DNase solution (i.e.,100 microliters of solution containing 8U RNase-free DNase/75U RNasin at 37^o C for 30 min).


2. Inactivate enzyme, wash slides and proceed with protocol

Appendix 5 - RT-PCR

Materials

Method

1. Add 100 microliter reaction mixture containing appropriate number of units of reverse transcriptase in 1X RT buffer and incubate at an appropriate temperature (i.e. 37-42^o C) for 30 minutes to 1 hour


2. Wash with 1X SSC


3. Inactivate room temperature by heating to 95^o C for 5 minutes


4. Wash five times with 1X SSC


5. Add 100 microliters of PCR amplification solution


6. Cover the slide with a glass coverslip and seal it with nail polish to prevent evaporation and reagent loss


7. Allow nail polish to harden for 5 minutes


8. Proceed with amplification per protocol (specific for the experiment)


9. Remove coverslip with a scalpel and scratch out remaining nail polish with a razor blade


10. Wash slides in 0.1M Tris-HCl, pH 7.4 twice for 5 minutes each


11. Dip slides in 100 % ethanol for 10 minutes to immobilize amplified products


12. Rehydrate in 80 % ethanol and wash in 0.1M Tris-HCl, pH 7.4, twice for 5 minutes


13. Proceed with detection

Appendix 6 - Polymerase chain reaction

Materials

Method

1. Remove slides from ethanol and rehydrate in water
   Place approximately 30-50 microliters of amplification cocktail onto the slide


2. Cover slide with a glass coverslip and seal with nail polish to prevent evaporation and reagent loss.


3. Allow nail polish to harden for 5 minutes


4. Proceed with amplification protocol (specific for the experiment)

Materials.

Method

1. Wash briefly in 2XSSC at 40^o C x 10 minutes


2. Wash in PBS x 2 minutes and then water


3. Dip slides in 100 % ethanol for 10 minutes to immobilize amplified products


4. Rehydrate in 80 % ethanol and wash in 0.1M Tris-HCl, pH 7.4, twice for 5 minutes each

Appendix 8 - Indirect in situ PCR Protocol

Materials

(*) = 500 mM KCl + 100 mM Tris-HCl pH 8.3

Method

1. Remove coverslip with a scalpel and scratch out remaining nail polish with a razor blade


2. Wash slides in 2X SSC at 37^o C for 5 minutes


3. Fix sections in 2 % paraformaldehyde for 10 minutes


4. Washed in PBS twice for 2 minutes each


5. Dehydrate in 100 % ethanol for 10 minutes and air dry


6. Add 20-30 microliter of hybridization solution containing 2X SSC, 50 % deionized formamide, 10 % dextran sulfate, 250 micrograms/milliliter salmon sperm DNA, and 5 nanograms of probe/slide


7. Apply coverslip


8. Denature target and probe DNA simultaneously in a water bath at 96^o C for 5 minutes


9. Hybridize 8 hours to overnight in a humidified chamber at 42^o C


10. Displace coverslip by dipping slide in 4X SSC at room temperature for 1-2 minutes


11. Wash at high stringency in 2X SSC at room temperature and then in 2 X SSC, 0.5X SSC and 0.1X SSC baths at 42^o C

Appendix 9 - Detection methods

Method

1. Rinse slides and place them in PBS x 5 minutes


2. a) For Biotin, add 100 microliters of the appropriate dilution of alkaline phosphatase-conjugated avidin or Streptavidin-Biotin complex b) For Digoxigenin, add reccommended dilution of Fab'-antidigoxigenin-alkaline-phosphatase


3. Rinse in 1X PBS twice for 5 minutes each


4. Apply appropriate substrate following vendor instructions


5. Stop reaction by transfering slides to a jar containing deionized water twice for 5 minutes each


6. Counterstain with modified Mayer's Hematoxylin x 30 seconds


7. Wash slides in running water x 10 minutes


8. Apply mounting media and coverslip.