John M. S. Bartlett.pdf - Bio-Nica.info

438 Wiedorn and Goldmann
2. Incubate 4 min at 95°C.
3. Incubate 2 × 5 min in 0.1 M Tris-HCl, 0.1 M NaCl, pH 7.5, in a Coplin jar.
3.8. DNAse/RNAse Treatment
If destruction of RNAse is necessary too (for example to generate negative controls),
a combined DNAse/RNAse treatment is recommended. Proceed as mentioned under
Subheading 3.7., but use the DNAse/RNAse solution.
3.9. Quenching
If Peroxidase/AEC or -/DAB systems are used during the detection then quenching
is required to inactivate endogenous peroxidase.
1. Incubate 30 to 45 min in 100 µL of 0.6% hydrogen peroxide (see Note 13).
2. Incubate 1 min in 50% ETOH.
3. Incubate 1 min in 70% ETOH.
4. Incubate 1 min in 90% ETOH.
5. Dry Slides for 5 min at 37°C (on the thermocycler).
3.10. Reverse Transcription
For all RT and IS-PCR, the Omnislide and MISHA thermocyclers have to be set to
simulated slide control and calibration factor 100.
If you are investigating DNA targets, then proceed to Subheading 3.11.
1. Incubate for 60 min at 39°C in 15 to 20 µL of RT-buffer per sample on the thermocycler.
Cover sample with coverslips and seal with PattexSupermatic200Plus (see Note 14).
2. Stop reaction by incubation at 92°C for 10 min.
3. Remove coverslips (see Note 15) and discard reaction mixture.
3.11. IS-PCR
Use aerosol resistant pipet tips for preparing the PCR.
1. Prepare PCR mix without Taq-Polymerase in Eppendorf tubes.
2. Heat PCR mix to 80°C.
3. Heat slides to 80°C on the thermocycler.
4. Add Taq-Polymerase to PCR mix and vortex briefly.
5. Pipet 16 µL of the PCR mix onto the specimen.
6. Immediately cover with a coverslip using a forceps and seal with PattexSupermatic
200Plus.
7. Heat forceps in a laboratory gas burner before proceeding to the next sample to avoid
cross contamination.
8. Repeat steps 5–7 for each specimen.
9. Start the PCR program.
10. Remove coverslips (see Note 15) and discard reaction mixture.
The PCR protocol for IL6 is composed of one cycle at 92°C for 5 min, 32 cycles each
with 1 min denaturation step at 92°C, 1 min annealing step at 56°C, 2 min extension
step at 72°C. The program is finished by an additional extension for 7 min at 72°C.
3.12. ISH
This step is only necessary if indirect IS-PCR is performed. Otherwise proceed to
Subheading 3.13. All reactions are performed on the thermocycler.