Non-radioactive in situ hybridization using biotinylated oligodeoxynucleotides and a detection protocol involving monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase system was employed for quantitation by image analysis. Calibrations of the image analysis system with neutral density filters revealed that the grey levels recorded were strongly linearly correlated to the absorbance (r2 = 0.97) in the range studied in tissue specimens (0-0.8 optical density or absorbance units). Several methodological parameters, including light source stability, section thickness, probe concentration and development time were initially optimized. Model systems revealed that the grey level measured varied linearly with the logarithm of the target concentration. Moreover, histophysiological studies on adrenalectomized and sham-operated rats documented that previous biochemical data on an 8- to 10-fold increase in anterior lobe proopiomelanocortin (POMC) mRNA levels 8 days after adrenalectomy are accounted for both by an increased ACTH cell concentration and content of POMC mRNA, as well as by increases in ACTH cell sizes and cell numbers. Also in agreement with biochemical data, image analysis did not reveal significant differences between OD's of melanotrophs in adrenalectomized and sham-operated animals. To our knowledge, these data are the first to document that non-radioactive in situ hybridization can be employed for relative quantitation. A particular advantage of this approach is the good morphological definition which permits parallel analyses of densitometric values, cell sizes and cell areas/cell numbers.

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