Sponge constructs for C2Orf41 and STARD13 were constructed by annealing the respective oligonucleotides together (Additional file 3, Table S2), top strand oligo (200 μM): 5 μl bottom strand oligo (200 μM): 5 μl, 10 × Oligo Annealing Buffer (BLOCK-iT kit*, Life Technologies, Carlsbad, California, USA): 2 μl, DNase/RNase-free water: 8 μl.
Membranes were blocked for 4 hr with blocking buffer (blocking reagent, Sigma-Genosys) and subsequently incubated with CtpB.
All the solutions including cell lysis buffer, blocking buffer and wash buffer were from this kit and the experiment was performed following the manufacturer's instructions.
Anti-FLAG® M2 agarose matrix had been previously equilibrated in TKM buffer, blocked with 1 mg/ml BSA for 30 min and washed again with TKM buffer.
Sections were boiled in citrate buffer, blocked with 1% BSA and 3% non-immune goat serum and incubated overnight with a rabbit polyclonal antibody to RXFP1 (Phoenix Pharmaceuticals, Burlingame, CA, USA) diluted 1 200 in blocking buffer.
After a minimum of 2 hours equilibration with the respective restriction enzyme buffer, blocks were incubated with restriction enzymes for 48 hours at 37°C.
Wells were washed thrice with washing buffer, blocked with 0.2 ml DAPI at 1 μg/ml for 5 min, and washed twice more.
Ten microliters of the probe hybridization mix (LSI® buffer, blocking DNA, labeled probes) were added to the specimens, slides were coverslipped, and sealed with rubber cement.
