Randomization is performed by the NTNU web-CRF solution with separate blocks and stratification to age and gender for each collaborating hospital.
The sections were microwaved in EDTA solution, cooled, and incubated with blocking solution (TBS-BSA 3%-Tween 0.1%) then with the primary antibody for 1 h at 37°C.
The membrane was first pre-blocked with blocking solution (PBS containing 2% BSA, 0.2% Tween-20, 0.05% sodium azide) for 1 hr at room temperature, followed by incubation with antibodies in blocking solution for 2 hr at room temperature.
Cells were cultured on cover slips, fixed with 4% paraformaldehyde (PFA) solution, blocked with 1 m M glycine (pH 7.5) and permeabilised for 4 min in 0.1% Triton.
Then, the membrane was washed with 1× PBST solution and blocked with 5× skim-milk blocking solution for 1 h.
After blocking, specimens were incubated with the primary antibody for 2 hours at RT in blocking solution with anti-calbindin (Synaptic Systems, Goettingen, Germany).
Sections of formalin-fixed paraffin-embedded blocks of NSCLC tumour samples and transgenic control cell pellets were deparaffinised in xylene, dehydrated in a graded ethanol series, and incubated with blocking solution (Peroxid Block, Zytomed Systems, Berlin, Germany).
This incubation occurred in blocking solution with 1% skimmed milk (incubation solution).
Sections were incubated overnight at 4 °C in the blocking solution with appropriate primary antibodies.
Sections were incubated with the Mouse-on-Mouse (MOM) kit blocking solution with avidin for 1 h.
