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

436 Wiedorn and Goldmann
41. Proteinase K solution: Proteinase K (Roche Diagnostic GmbH, Mannheim, Germany;
250 µg/mL), 100 mM Tris-HCl and 50 mM EDTA.
42. 10× Target Retrieval (Dako, Hamburg, Germany, Cat-No. S1700).
43. DNAse solution: 40 mM Tris-HCl, pH 7.4, 6 mM MgCl 2 , 2 mM CaCl 2 , and 1 U/µL RNAse
free DNAse (Roche Diagnostic GmbH, Mannheim, Germany).
44. IL6 PCR mix: 10 mM Tris-HCl, pH 9.2, 50 mM KCl, 3 mM MgCl 2 , 0.2 mM dNTPs
(Roche Diagnostic GmbH, Mannheim, Germany); 0.1% BSA (Roche Diagnostic GmbH,
Mannheim, Germany); 10 µM DIG-11-dUTP (Roche Diagnostic GmbH, Mannheim,
Germany) (see Note 3); 5 U/sample Taq-Polymerase (Roche Diagnostic GmbH, Mannheim,
Germany); and 0.4 µM of sense and antisense primer.
45. Sequence of IL6 primers (see Note 4): sense primer: 5′ CTTCTCCACAAGCGCCTTC-3′;
antisense primer: 3′ CTAAGTTACTCCTCTGAACGG-5′.
46. A typical hybridization mix is composed of (exact compositions depends on the probe in
use): 2× up to 5× SSC; up to 50% formamide; 5% up to 10% dextran sulfate (see Note 5);
0.1% SDS; 0.1% BSA; 1% up to 2% blocking solution; 250 µg/ mL fish sperm DNA
(Roche Diagnostic GmbH, Mannheim, Germany); 1–10 ng/µL hybridization probe.
Additionally for RT-IS-PCR:
47. DNAse/RNAse solution: 40 mM Tris-HCl, pH 7.4, 6 mM MgCl 2 , 2 mM CaCl 2 , 1 U/µL
RNAse free DNAse (Roche Diagnostic GmbH, Mannheim, Germany), and500 µg/mL
RNAse (Roche Diagnostic GmbH, Mannheim, Germany).
48. Reverse transcription buffer (RT-buffer): 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM
MgCl 2 , 0.5 mM dNTPs (Roche Diagnostic GmbH, Mannheim, Germany), 10 mM DTT
(GibcoBRL, Karlsruhe, Germany), 1 U/µL RNAsin (Promega, Mannheim, Germany),
2 U/µL M-MLV-RT (GibcoBRL, Karlsruhe, Germany) (see Note 6), 0.1 µg/µL Random
Hexamers (Roche Diagnostic GmbH, Mannheim, Germany).
3. Methods
If not otherwise mentioned, all incubations are performed at ambient temperature
(20°C). Incubations on the thermocycler are performed using the humidity chamber.
Samples are processed without coverslips and sealing if not otherwise mentioned.
3.1. Prevention of DNAse and RNAse Contamination
1. Incubate all slides, coverslips and microtome blades for 12 h at 220°C to inactivate DNAse
and RNAse (see Notes 7 and 8).
2. If RNA detection is required, include 0.1% DEPC in all solutions and incubate overnight.
3. Autoclave solutions for 20 min at 1.2 bar (15 psi) at 121°C.
3.2. Fixation
Incubate tissues for 24 h in 5% buffered formalin (for detection of DNA) or 4%
buffered paraformaldehyde (for detection of mRNA) (see Note 9). With cell suspensions
and cytospins a 30 min incubation is usually sufficient.
3.3. Paraffin Embedding
The distilled water used to dilute ETOH should be autoclaved and prepared with
DEPC-treated water to ensure the absence of DNAse and RNAse.
1. Incubate tissue for 30–60 min in 70% ETOH.
2. Incubate tissue for 30–60 min in 80% ETOH.