title: In situ hybridization histochemistry quantification: automatic count on single cell in digital image.
abstract: It is extremely useful in investigations of the central nervous system (CNS) to measure mRNA expression in cells by in situ hybridization. However, this approach is limited by the difficulties of a reliable quantitative evaluation. In the present paper we describe a method for quantifying radioactive hybrids on individual cells by a silver grain count in digital images. Quantification is based on the real size and grey level of a single grain obtained by computerized microscope image analysis (IBAS 2, Kontron-Zeiss, PC 286). The program provides for automatic identification of cell area, the portion occupied by grains and their grey level. The number of grains per cell results from a mathematical function integrating these parameters with a 'density factor'. This factor is introduced to better estimate the number of grains when there is overlapping. The method was tested by measuring the expression of preproNPY (pp-NPY) mRNA in rat dentate gyrus and preproSomatostatin (pp-SOM) mRNA in frontal cerebral cortex of control and colchicine-treated rats. Colchicine did not modify the number of pp-SOM mRNA-positive cells but reduced the expression per cell. These results confirm the advantages of our method to quantify a wide range of silver grains (10-5000) and improves the sensitivity of in situ hybridization. With this support the in situ hybridization technique could be considered a real quantitative method for measuring small alterations in neuronal function.
authors.affiliation: Department of Geriatric Neuropsychiatry, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Masseroli
authors.forename: A
authors.initials: A
authors.lastname: Bollea
authors.forename: C
authors.initials: C
authors.lastname: Bendotti
authors.forename: G
authors.initials: G
authors.lastname: Forloni
mesh_terms: D000703:Analog-Digital Conversion; D000818:Animals; D048429:Cell Size; D003720:Densitometry; D015870:Gene Expression; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization; D008297:Male; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D009478:Neuropeptide Y / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D012984:Software
chemical_list: D009419:Nerve Tissue Proteins; D009478:Neuropeptide Y; D012333:RNA, Messenger