Phiv, Histocytologie and plant cell imaging platform

In situ hybridization

In situ hybridization is a powerful technique used to visualize the ARN messengers in the tissues and in the cells, thus allowing a spatio-temporal analysis of gene expression in animal and plant tissues. It is a key step in the functional gene analysis.
It consists in creating hydrogen bonds between the nitrogenized bases of the sequence of a nucleic acid carrying a marker (a probe) and the complementary sequence present in tissues, thus generating a detectable signal under the microscope.

It is a long and delicate technique. PHIV team has a long experience in in situ hybridization acquired on a great diversity of organs and species (on the model plants Arabidopsis thaliana, Medicago truncatula, Oriza sativa but also on tropical species, cacao-tree, palm tree, hevea etc…) and on various types of genes (slightly expressed genes, transcription factors, ion transporters…)
The various steps of the experiment (preparation of the samples, slicing, synthesis and priming of the probes, hybridization itself and observations under the microscope) are delicate and difficult to standardize. With our background on plants, we propose several protocols in order to adapt to tissues and genes. These adaptations relate as much to the preparation of the samples and the synthesis of the probes that on the hybridization conditions and the signal revelation. In certain cases, we perform a secondary revelation in fluorescence (secondary antibodies combined with a fluorochrome) in order to increase the strength and the precision of the signal.
The images are acquired with an epifluorescence or confocal microscope with spectral analysis in order to eliminate the autofluorescence of the tissues (walls, phenolic compounds, etc…) and to validate the signal obtained.

Localization of ARN messengers of ferritin by HIS in a leaf of Arabidopsis thaliana (Alexa 488)

 


AtFer1


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Positive

Spectrale deconvolution

 

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