Modifications included increasing the amount of starting mRNA to 20 μg, the addition of T4gene32 protein during first strand synthesis [ 82], the use of the plasmid pDNR-Lib (Clontech, Mountain View, CA, USA) for cDNA cloning, and the omission of radioactive labelling to monitor first strand synthesis.
Probe specificity was checked by substitution of the respective antisense cRNA probes with an equivalent amount of labeled sense cRNA probe and by omission of cRNA probes.
The survival of labelled eGFP-5T33 cells in vitro is comparable to or better than that of [In]oxinate3-labelled cells and not significantly different to that of control cells treated identically except for omission of radioactivity.
For labeling, omission of the primary antibody served as a negative control.
In addition, omission of the incubation step with labeled lectin from routine processing enabled us to exclude lectin-independent signal generation.
Controls for specificity of immunohistochemical labeling included omission of the primary antibody and use of preimmune normal mouse and rabbit serum as previously described [26], [52] [54].
For example, it was agreed that if a prescriber specified an indication on the prescription (such as omeprazole 'for oesophagitis'), then omission of this indication from the dispensing label was a labelling error.
Specificity of labelling and absence of signal crossover was excluded in all cases by examination of single labelled control samples, including labelling controls with omission of primary antibody.
Control experiments included incubation of sections or cells with non-immune isotypic control antibodies or the omission of primary antibodies followed by the appropriate labeled secondary antibodies.
A Western immunoblot was also prepared from identical samples with the omission of the proteinase K digestion step and labelled with antibody P4.
The immunoreactive signals were specific for Reelin, since control stainings with isotypic IgGs (mouse anti-human CD45) or the omission of primary antibodies did not yield any specific labeling.
