PMID: 7536187Jan 1, 1995Paper

The effects of varying key steps in the non-radioactive in situ hybridization protocol: a quantitative study

The Histochemical Journal
Y Guiot, J Rahier

Abstract

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 micrograms 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 microns before slowly decreasing. A weak paraformaldehyde post-fixation (0.4% for 20 min) gives best ...Continue Reading

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Citations

Mar 1, 2007·Acta Histochemica Et Cytochemica·Takashi KogamiJunzo Sasaki
Mar 1, 1997·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·A JonkerA F Moorman
May 20, 2004·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Touji KimuraJunzo Sasaki
Oct 5, 2002·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Erik Hrabovszky, Sandra L Petersen
Jun 17, 2000·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·S Vizi, K Gulya
Apr 28, 1999·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·D A Bessert, R P Skoff
Mar 20, 1999·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·H YangN Chaudhari
Feb 12, 1998·Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B·Y Guiot, J Rahier
Nov 15, 2002·European Journal of Haematology·I TheateUNKNOWN Groupe d'Etude des Lymphomes de l'Adulte (GELA)
Oct 25, 2003·Journal of Neuropathology and Experimental Neurology·Rita Machaalani, Karen A Waters

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