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

Direct and Indirect In Situ PCR 439
1. Prepare fresh hybridization mix without fish sperm DNA.
2. Denature fish sperm DNA for 10 min at 100°C in a water bath.
3. Cool fish sperm DNA on ice for 2 min.
4. Add fish sperm DNA to the hybridization mix.
5. Pipet 16 µL of the hybridization mix onto each sample.
6. Immediately cover with a coverslip using a forceps and seal with PattexSupermatic200Plus
(see Note 14).
7. Heat forceps in a laboratory gas burner before proceeding to the next sample to avoid
cross contamination.
8. Incubate 10 min at 95°C.
9. Incubate 1 to 16 h between 30 to 65°C (see Note 16).
10. Remove coverslips (see Note 15) and discard reaction mixture.
3.13. Washing and Detection
1. Incubate 2 × 5 min in 0.1× SSC at 20°C (see Note 17).
2. Incubate 10 min in 0.1× SSC at 45°C in the Wash Module (see Note 17).
3. Incubate 1 min in 1× washing buffer (DIG Wash and Block buffer Set, Roche Diagnostic
GmbH, Mannheim, Germany).
4. Incubate 60 min in 1× blocking solution at 37°C.
5. Add 100 µL of 1250 diluted Anti-DIG-AP-conjugate per sample and incubate 60 min
at 37°C on the thermocycler.
6. Incubate 2 × 5 min in 1× washing buffer at 20°C.
7. Incubate 2 × 5 min in 1× detection buffer at 20°C.
8. Incubate in 100 µL per sample freshly made substrate solution (up to 12 h) in the
dark. Substrate solution is composed of (per mL): 4.5 µL NBT, 3.5 µL BCIP, 992 µL
of detection buffer (all reagents Roche Diagnostic GmbH, Mannheim, Germany) (see
Note 18).
9. Counterstain 10 min with nuclear fast red or eosin at 20°C (intensity of counterstain may
be adjusted by reducing or prolonging incubation period).
10. Mount with Permount SP15-500 (Fisher Scientific).
3.14. Controls
Several controls have to be performed to ensure the specificity of the results.
1. Omission of RT (should result in cells showing no signal).
2. Omission of Taq-Polymerase (should result in cells showing no signal).
3. Omission of primers (should result in cells showing no signal).
4. RT IS-PCR of a housekeeping gene, such as GAPDH, or actin to ensure that the reverse
transcription and PCR was successful because GAPDH should be demonstrable in each
cell. Furthermore, this should indicate if the permeabilization was successful.
5. PCR of Alu-repeats to ensure that PCR and permeabilization was successful.
6. Omission of the Anti-Dig-POD or Anti-DIG-AP to detect endogenous enzyme activity.
7. Tissues that are definitively negative for the sequence under investigation. If such tissues
are not available, the control samples may be treated either with DNAse, RNAse or both.
8. Tissues that are definitively positive for the sequence under investigation.
Furthermore, at least in the phase of establishing a new PCR protocol for new
primers, all IS-PCRs should be controlled by simultaneous solution-phase PCRs on
serial sections of the same samples.