THE EFFECTS OF VARYING KEY STEPS IN THE NONRADIOACTIVE IN-SITU HYBRIDIZATION PROTOCOL - A QUANTITATIVE STUDY

In situ hybridization represents a major advance in the study of gene expression and, thus, in the evaluation of cellular function in histological sections. The availability of oligonucleotide probes labelled with biotin and sensitive immunohistochemical detection systems makes the study of different types of mRNA by in situ hybridization easier. However, a large number of protocols have been reported, which is sometimes confusing. The present study analyses quantitatively the influence of each important step of in situ hybridization on the staining intensity of rat proinsulin mRNA. The aim was to optimize technical conditions, to make the method sensitive and to evaluate its reproducibility for proinsulin mRNA detection and measurements. The duration of fixation and the digestion have an important impact on the results. The optimal digestion time depends on the fixation. With a digestion of 30 mu g ml(-1) proteinase K for 12 min at 37 degrees C, the optimal fixation time was 24 h. Section thickness also influences the staining intensity. The intensity of the staining increases as the section thickness increases from 3 to 5 mu m before slowly decreasing. A weak paraformaldehyde post-fixation (0.4% for 20 min) gives best results in comparison to a stronger post-fixation (4% for to min). An increase of probe concentration leads to a higher specific labelling, reaching a plateau at 800 ng ml(-1). Hybridization temperature (37-42 degrees C) exerts little influence. However, the temperature of the washes and the immunodetection system have a major effect on the intensity of labelling. Quantification has permitted the evaluation of the influence of each key for optimizing and standardizing the non-radioactive in situ hybridization protocol. In these well-defined conditions, the intra- and inter-assay coefficient of variation remains lower than 6% and thus the method can be used to quantify the content of proinsulin mRNA or other specific mRNAs in experimental and pathological conditions.