In situ hybridization histochemistry quantification: automatic count on single cell in digital image

M. Masseroli, A. Bollea, C. Bendotti, G. Forloni

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)93-103
Number of pages11
JournalJournal of Neuroscience Methods
Volume47
Issue number1-2
DOIs
Publication statusPublished - 1993

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Keywords

  • Autoradiography
  • Central nervous system
  • Computerized microscope image analysis
  • mRNA expression
  • PreproNPY mRNA
  • Somatostatin

ASJC Scopus subject areas

  • Neuroscience(all)

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