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

RT In Situ PCR 447
Fig. 2. Schematic of the appropriate controls, how they are achieved, and the expected results
that indicate a successful reaction.
8. Primers (see Subheading 4.6.). The following primer pair yields a 295-bp amplicon for rat
TNF mRNA: 5-TACTGAACTTCGGGGTGATCGGTCC-3 and 5-CAGCCTTGTCCCTT
GAAGAGAACC-3.
9. M-MLV RT enzyme (Gibco/BRL).
10. Reverse transcription buffer: 75 mM KCl, 3 mM MgCl 2 , 50 mM Tris-HCl, 0.1 M DTT
(pH 8.3), 1 µM antisense primer or 25 µg/mL oligo-(dT) 12–18 primer, and 2500 U/mL
M-MLV RT enzyme.
11. Taq DNA polymerase (Gibco/BRL).
12. PCR buffer: 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 4.5 mM MgCl 2 , 80 µM mixed dNTP,
16 µM digoxigenin-11-dUTP, 1.2 µM each primer, and 120 U/mL Taq polymerase.
13. Digoxigenin-11-dUTP (Boehringer Mannheim).
14. Anti-Digoxigenin antibody, alkaline phosphatase conjugated (Boehringer Mannheim).
15. Wash buffer 1: 0.1 M TRIS, 0.15 M NaCl, pH 7.5.
16. Wash buffer 2: 0.1 M TRIS, 0.15 M NaCl, pH 9.5.
17. Chromagen: 4-nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indoyl phosphate
(BCIP) (Boehringer Mannheim).
18. Substrate solution : 10µL of NBT, 7.5 µL of BCIP in 2 mL of buffer 2.
19. Water, di-ethyl pyrocarbonate (DEPC) treated, RNase free.
20. Silane-coated glass slides.
21. Thermal cycling PCR machine or dedicated in situ PCR machine.
3. Methods
3.1. Cell Fixation
1. Cells collected from tissue culture flasks, blood, or other sources are collected in a 15-mL
polypropylene tube.