TY - JOUR
T1 - In situ hybridization histochemistry quantification
T2 - automatic count on single cell in digital image
AU - Masseroli, M.
AU - Bollea, A.
AU - Bendotti, C.
AU - Forloni, G.
PY - 1993
Y1 - 1993
N2 - 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.
AB - 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.
KW - Autoradiography
KW - Central nervous system
KW - Computerized microscope image analysis
KW - mRNA expression
KW - PreproNPY mRNA
KW - Somatostatin
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U2 - 10.1016/0165-0270(93)90025-M
DO - 10.1016/0165-0270(93)90025-M
M3 - Article
C2 - 8321017
AN - SCOPUS:0027215791
VL - 47
SP - 93
EP - 103
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
IS - 1-2
ER -