Phiv, Histocytologie and plant cell imaging platform


Plant protein Immunolocalization

The Immunocytochemistry allows the localization of tissue or cellular proteins in plant organs. It utilizes the complexation antigen-antibody via specific antibodies directed against the requested protein.

We propose a technique known as indirect, the marker (enzyme or fluorochrome) being combined with a secondary antibody. Within the framework of this technique, we developed several protocols in order to adapt to the great diversity of plant tissues. These protocols can be used on samples imbedded in paraffin or resin, or on fresh tissues (entire organs or sliced obtained with a vibratome), which can result in important time saving.
The protocol variations relate to the preparation of the samples, in particular the fixing, a very complex and crucial step, dehydration and the use of fluorochromes for protein detection to improving the specificity of the signal. The confocal and mutiphoton microscope of the laboratory is equipped with a spectral analyser so discriminating the specific signal out of the autofluorescence (cellular wall and many phenolic compounds present in plant tissues). 3D images can be obtained from intact organs or from thick slices.
Finally the use of different fluorescence markers makes possible to localize different proteins on the same slice by using antibodies of different origin or different isotypic nature, bound to distinct fluorochromes with distant emission peaks.

Defensine immunolocalization in a leaf ofArabidopsis halleri


Transverse leaf section (photo de Marie Mirouze)

Trichome (photo de Marie Mirouze)

Vibratome-made slices stained with Alexa-488-combined antibody.

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