Combined IF/FISH

Fluorescent in situ hybridizations were performed using the Tyramide amplification method (TSA) following the protocol described by Krabichler et al.⁷³. At the beginning of the procedure, sections were incubated in a 3% hydrogene peroxide solution in 10% methanol, to inactivate endogenous peroxide. Also, after washing the sections with A and B solutions, two additional 5 min washes were performed using TNT (100 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20 in filtered H₂O). Then, sections were incubated at room temperature for 3 h in blocking solution (Blocking Buffer Reagent 1%, Heat Inactivated Horse Serum (HINHS) 1% in TNT). Finally, sections were incubated overnight in a new blocking solution containing the anti-Digoxigenin-POD, Fab fragment (Roche) (1/300) at 4ºC. The following day, sections were washed during 30 min at room temperature in TNT, followed by a 10 min wash in 0.05 M Borate Buffer, pH 8.5. Then, sections were incubated for 1 h, at room temperature in darkness, in a Biotin-tyramide (IRIS Biotech GmbH, Marktredwitz, Germany) solution (0.001% Biotin-tyramide and 0.0015% H2O2 in 0.05 M Borate, pH 8.5). After 3 washes in PBS, fluorescent label was obtained by 2-h incubation with Streptavidin-Alexa-546 (1/500) in PBS and 0,25% Tween 20.

IHC and FISH combined in the same sections was obtained by performing first the FISH protocol to continue with the immunoreaction as described above.