Fast red tr salt solution

Tumor samples were fixed in 10% buffered formaldehyde and then decalcified in 4% EDTA 0.2% paraformaldehyde (PFA, pH 7.4) buffer for 4 weeks. After embedding in paraffin wax, 5 μm-thick sections were stained with Haematoxylin–Eosin–Safran (HES). Human nucleus detection was performed using a digoxin-labeled human locked nucleic acid Alu probe (Exiqon, Vedbaek, Denmark) as described previously [10] (link). Briefly, 70 nM Alu was hybridized on histological sections following DNA denaturation, in a buffer containing 4 X SSC (S6639, Sigma Aldrich, St. Quentin Fallavier, France), 50% deionized formamide, 1 X Denhardt's solution, 5% dextran sulfate and 100 µg/mL Salmon sperm DNA, for 19 h at 56 °C. Finally, the Alu probe was detected by peroxidase-based immunohisto-chemical procedure. For Tartrate-Resistant Acid Phosphatase (TRAP) detection slides were incubated 1 h in a 1 mg ml⁻¹ naphthol AS-TR phosphate, 60 mmol l⁻¹N,N-dimethylformamide, 100 mmol l⁻¹ sodium tartrate and 1 mg ml⁻¹ Fast Red TR salt solution (Sigma Aldrich, Saint Quentin Fallavier, France) and counterstained with haematoxylin. EGFP detection was observed on frozen sections with fluorescence microscopy directly or after immunostaining using mouse monoclonal to GFP primary antibody (Abcam, Paris, France) and Alexa Fluor 594 goat anti-mouse IgG (Life Technologies, Saint-Aubin, France).