During hydrosilylation a small excess of diethoxy methyl)silane was reacted using hydrogen hexachloroplatinate (H2PtCl6·5H2O) as the catalyst.
For triple labelings, NPY cRNA was reacted using biotin-RNA labeling mix, 5-HT2C cRNA with fluorescein-RNA labeling mix and 5-HT1A cRNA with DIG-RNA labeling mix.
For TMV-iAlk, 25 molar excess of propargylamine per coat protein was reacted using EDC coupling with 45 molar excess of both EDC and HOBt at a final concentration of 2 mg/mL TMV in HEPES buffer.
In brief, 25 molar excess of propargylamine per coat protein was reacted using EDC coupling overnight to produce TMV-iAlk, 35 equiv of ethynylaniline diazonium salt was reacted for 30 min to yield TMV-eAlk, and 10 molar excess of NHS-alkyne was reacted overnight with TMVLys to form TMVLys-eAlk.
Alternatively, low grade substrates and/or non-acylglycerol lipids can be reacted using Brönsted acids.
The DNA backbone is subsequently cleaved at the sites where the bases have been reacted, using hot piperidine.
Procedures for hapten labeling of cRNA probes were done as follows: for single labelings, cRNAs were reacted using biotin-, fluorescein- or digoxigenin (DIG -RNA labeling mixes (all Roche, Mannheim, Germany).
PNA monomers and AEEA-COOH linker were reacted using the following conditions: monomer building block (8 eq. in NMP 0.4 M), HATU (8 eq. in DMF 0.4 M), and DIPEA (8 eq)./lutidine (12 eq).
A number of different nucleobase carboxylic acids and various amines were reacted using alternative coupling reagents such as HBTU [2-(1 H-benzotriazol-1-yl -1,1,3,3-tetramethyluronium H-benzotriazol-1-yl -1,1,3,3-tetramethyluroniumarbodiimide] H-benzotriazol-1-yl -1,1,3,3-tetramethyluroniuminked compounds B(i–viii) given in Figure 1.
The cDNA was then reacted using Gene Navigator cDNA Array Filter-human cancer (Toyobo, Osaka, Japan) and subjected to DNA array analysis using a Fluor-S Multi Imager Bio-Rad Laboratoriess, Hercules, CA, USA).
Sections were then reacted using the avidin-biotin complex method (Vector Laboratories, CA, USA) for 30 min, followed by washing in biotinylated IgG antibody (Vector Laboratories) for 30 min. Afterwards, sections were stained using a 3,3'-diaminobenzidine, 0.0045% hydrogen peroxide solution and placed on a glass slide to keep dry.
