After checking the quantity and quality of aRNA by spectrophotometry using NanoDrop™ 1000 (Thermo Scientific) and the quality subsequent labelling, 4 μg of labelled aRNA was hybridized to the array according to the manufacturer's recommendations [ 24].
Antibody premodification and subsequent labeling with 89Zr were performed as described previously, using desferal [Df] (desferrioxamine B, Novartis Pharma BV, Arnhem, The Netherlands) as the chelate [33] (see Additional file 1).
Instead, the bacteria S. typhimurium were firstly incubated with the free biotin-labeled aptamer that could result in mass molecules bounding to the bacteria surface, which would facilitate the subsequent labeling of SA-FSiNPs.
It can effectively be achieved by: i) the introduction of a longer polyethylene glycol chain spacer, or ii) the presence of a positive charge, known to favour renal elimination, or iii) attachment of a chelating agent and subsequent labelling with a radiometal.
All subsequent labelling was carried out in blocking buffer (primary antibody 1∶500 and secondary 1∶250).
The GFP expressing cells can be purified by FACS technology to contain nearly 90% photoreceptors, as assessed with subsequent labeling with rod and cone markers.
Transfection was performed using Lipofectamine 2000 (Invitrogen) as per manufacturer's instructions, and subsequent labelling and imaging was carried out between 24 40 h post-transfection.
After 18 hours at 37°C and 5% CO2, BrdU labeling solution was added to each well and the subsequent labeling and detection were carried out following the manufacturer's protocols.
For microarray hybridizations, UTP-amino allyls were integrated during the second round of in vitro transcription, for subsequent labelling with dyes Cy3 or Cy5 (Amino Allyl MessageAmp II kit - Ambion).
These comprehensive CSF proteomics datasets were then used to create an accurate mass and time (AMT) tag database for subsequent label-free quantitative analysis of individual subject CSF samples.
