This section describes an abbreviated method for in situ PCR, specifically reverse transcriptase in situ PCR. Each of these experimental procedures is discussed in detail. This experiment is derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Reverse transcriptase in situ PCR assay
Principle
In Situ PCR Instrument, Coverslips, Incubator, Wet Box, RT-PCR Kit, Permount
Materials and Instruments
Tissue Samples and Cell Cultures Move I. Materials For more product details, please visit Aladdin Scientific website.
Nuclear Fast Red Formaldehyde buffer Assay solution DEPC-treated water PBS KH2P04 Xylene Ethanol Bovine serum albumin SSC NBT BCIP Color developer Pepsin DNA enzyme Taq DNA polymerase RNA enzyme inhibitor DNA enzyme buffer dUTP Digoxin-resistant coupler PCR primers
In Situ PCR Instrument Coverslip Incubator Wet Kit RT-PCR Kit Permount
1. Buffers and solutions
Nuclear Fast Red
10% (v/v) Formaldehyde Buffer (Polyscientific)
Assay solution (0.1 mol/L Tris-HCl, pH 9.5, 0.1 mol/L NaCl)
DEPC-treated water
Phosphate buffer solution (PBS) (137 mmol/LNaCl, 2.7 mmolKCl,10 mmol/LNa2 HPO4 )
2 mmol/L KH2P04, pH 7.0, for cell cultures
Xylene (for tissue samples)...
Ethanol
Bovine serum albumin (BSA), 20 g/L
SSC, 20X (3 mol/L NaCl, 0.3 mol/L sodium citrate, pH 7.0)
NBT/BCIP Color Developer (EnzoClinicalLabs)
2. Enzyme and enzyme buffer
Pepsin (2 mg/ml) (Enzo Diagnostics)
Add 20 mg of pepsin, 9.5 ml of H2O and 0.5 ml of 2 mol/L HCl.
DNA enzyme without RNAase (Boehringer Mannheim)
Taq DNA polymerase Gold (Applied Biosystems, Foster City, CA)
RNAase inhibitor (Applied Biosystems)
DNA enzyme buffer, 10X
Add 35ul of sodium acetate (3 mol/L, pH 3), 5ul of MgS04 (lmol/L) and 60ul of DEPC-treated H2O.
3. Nucleic acids and oligonucleotides
Digoxin-labeled dUTP (Boehringer Mannheim), 1 mmol/L
4. antibodies
Antidigoxigen inconjugate (Boehringer Mannheim)
Suitable PCR primers
5. Specialized equipment
Silanized carrier sheets (Ventana)
In situ PCR instrument (AppliedBiosystems)
Polypropylenesheets for coverslips (commercially available in autoclaved bags)
Incubator set at 37°C and 60°C
Wet boxes (lidded boxes lined with water-soaked paper towels)
6. Other
RT-PCR kit (AppliedBiosystems)
This kit includes buffers, nucleotides, RNAase inhibitors, manganese solution, rTthDNA polymerase, and control primers for RT-PCR.
Reagents and equipment for paraffin embedding and sectioning (for tissues).
Permount
7. Cells and tissues
Suitable tissue samples and cell cultures
Many reagents including proteases, buffers, washes, color developers, probes and re-staining agents are included in the kit for in situ hybridization. Many biological companies now sell the kit.
II. Methods
1. Preparation of tissue sections and cell samples
(1) Fix the materials in 10% formaldehyde buffer for 8~15 h, and then embed them in paraffin.
(2) In case of cell cultures, add PBS directly into the culture plate to wash the cells once. Then add 10% formaldehyde solution and fix overnight, then scrape the cells off the plate with a latex scraper. Wash the collected cells twice in DEPC-treated H2O, put them into a tabletop centrifuge and centrifuge at 2000r/min for 3 min. Resuspend the cells in 5 ml of DEPC-treated H20, take 50ul drops and add them on the carrier plate (optimal concentration: 2500~7500 cells), air-dry and store at room temperature.
(3) Take at least 3 4um thick paraffin tissue sections or 3 cell suspensions onto silanized slides and fix the material on the slides.
Silanization is essential for cell adhesion.
(4) Place the tissue sections in fresh xylene for 5 min to remove the paraffin, then place them in 100% ethanol for 5 min, remove, and air dry.
2. Protease treatment
(5) Place the prepared slices in pepsin solution and incubate at 37°C for an appropriate period of time.
Determine the duration of protease incubation in Tables 19-1 and 19-2. Proteases other than pepsin may also be used for digestion. See 19.2.1.2 Protease Digestion for more information.
(6) Place slides in DEPC-treated H20 for lmin to inactivate proteases, then place in 100% ethanol for 1min, remove, and dry.
This simple washing step is sufficient to remove/inactivate pepsin without heat inactivation.
3. DNA enzyme treatment (for reverse transcriptase in situ PCR) 
(7) Add lul of 10XDNAase buffer, lul of RNAase-free DNA enzyme (10 U/ul, 8 ul of DEPC-treated H20) to 2 of the 3 slices. RT/PCR buffer can also be used instead of 10XDNAase buffer.
(8) Cut a polypropylene sheet to the appropriate size for the sample material on the slide and cover with the added solution to prevent it from drying out. Place the slides in a wet box and incubate at 37°C.
(9) After overnight digestion, remove the cover slip and wash the carrier slice in DEPC-treated H20 for lmin, then in 100% ethanol, remove and dry.
4. Reverse transcription
(10) Take a sterile centrifuge tube and add the following reagents for RT/PCR.
EZ buffer (AppliedBiosystems) 10ul
Add 1.6ul of each of the 4 nucleotides (dATP, dCTP, dGTP and dTTP, each at a concentration of 10 mmol/L).
Bovine serum albumin (200 g/L) 1.6ul
DEPC-treated H20 15.lul
MnCl2 (or manganese acetate) solution (10 mmol/L) 12.4ul
Justice primer (20umol/L) 2.0ul
Antisense primer (20umol/L) 2.0ul
RNAase inhibitor 0.5ul
Digoxin-labeled dUTP (1mmol/L) 0.6ul
rTth 2.0ul
(11) Add the prepared RT/PCR reaction solution to one of the two DNAzyme-treated slide.
(12) To prevent drying out, attach the polypropylene cover slip to the slide with a small drop of nail polish, place the slide in an aluminum "boat" and cover it with sterile mineral oil. The slides were then placed on the block of the thermal cycler and held at 60°C for 30 min.
AppliedBiosystcms' Amplicover/Ampliclip method can be used as an alternative to mineral oil.
5. PCR 
(13) Place a sterile centrifuge tube on ice and add the following PCR reaction reagents.
Taq polymerase buffer (AppliedBiosystems) 5ul
dNTP solution (10 mmol/L for each nucleotide) 9.0ul
MgCl2, 25 mmol/L 8.0ul
Bovine serum albumin, 20 g/L 1.6ul
H2O 22.4ul
Justice primer (20umol/L) 2.0ul
Antisense primer (20umol/L) 2.0ul
Taq Polymerase Gold (10U/ul) 2.0ul
(14) The PCR reaction mixture was added to each sample and the following thermal cycling reaction was performed. 
(15) Remove the cover slip and nail polish from the slide. Wash in xylene for 5 min and place in 100% ethanol for 5 min, remove and air dry.
6. Detection
(16) Place the slide in 0.2XSSC containing 2 g/LBSA and wash at 60°C for 15 min (for reverse transcriptase in situ PCR).
For in situ hybridization PCR, if a full-length genomic probe of at least 80bp size is used, wash with the above wash solution. For hybridization with oligo probes smaller than 45bp, wash with 1XSSC containing 20 g/LBSA at 45°C for 10 min.
(17) Remove the residual wash solution and add 100ul of 0.lmol/L Tris-HCl, pH 7,5, 0.1 mol/L NaCl solution containing digoxin antibody (diluted 1:150) to each slide. incubate at 37°C for 30 min.
(18) Wash the slide with the assay solution for 1 min, then transfer it to the assay solution containing NBT/BCIP color developer and incubate at 37°C for 5-30 min, as described in the distributor's instructions (EnzoClinicalLabs). The incubation time can be shorter if the marker is directly incorporated into the amplified DNA, and longer if the probe is used. Place the slide under a microscope and terminate the reaction when a strong signal is seen. For in situ hybridization PCR, the probe-amplifier complex should be hybridized for 15 h; digoxigenin-labeled probes are recommended for this purpose. It is also important to use the longest probe possible (preferably 80-100bp), if possible. Since primer oligomerization does not normally occur in in situ hybridization PCRs, full-length probes containing primer segments can be used. Similarly, it is important to label probes using random extensions (Nuovo 1997). If shorter probes are used, they must not be shorter than 45bp and should be labeled using the 3' end method (Nuovo1997).
(19) Slides were washed in water for 1 min, re-stained with nuclearfastred for 5 min, washed again in water for 1 min, and then placed in 100% ethanol for lmin and xylene for lmin. the slides were sealed with Permount and observed under a microscope.