Phalloidin-Alexa Fluor 568 (Invitrogen A12380, diluted 1 50 in blocking buffer) and/or BrdU-FITC (Becton-Dickinson 347583, diluted 1 4 in blocking buffer) were applied for 30 min before final washes and mounting in Vector-shield containing 0.5 mg/ml DAPI (Sigma D9542).
After washing (3×20 min) in blocking buffer, sections were mounted in Mowiol (Calbiochem -containing 4% DABCalbiochem -containing
Blocking buffer was used to avoid non specific binding.
Each TMA block included 98 tissue cores and the total RCC material was mounted in six blocks.
The sample was mounted onto a polyvinylidene difluoride membrane and then incubated in blocking buffer (5% nonfat dry milk in Tris-buffered saline with 0.2% Tween 20) at room temperature.
After antibody incubation, coverslips were washed 3 4 times with blocking buffer and mounted using ProLong Gold Antifade with DAPI (Invitrogen).
The coverslips were subsequently incubated for 1 h in primary antibody followed by 40 min in secondary antibody, both diluted in blocking buffer, and finally mounted with VECTASHIELD mounting medium containing DAPI (4′,6-diamidino-2-phenylindole, Vector Laboratories, Bionordika, Stockholm, Sweden).
The piezoelectric transducer was mounted on the bottom of the platinum buffer rod.
Cells were then washed, incubated for 60 min with Alexa Fluor conjugate secondary antibodies, rinsed with blocking buffer and mounted on slides with DAPI containing ProLong Gold Antifade Reagent (Invitrogen).
