Biomedical research institute
     

    Equipments

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    Our facility has a complete equipment for histological studies.

    A first room is dedicated to the preparation of tissues for paraffin embedding with :

    -        a paraffin impregnation system (Sakura) : This automaton (VIP 5 junior, Tissue-Tek, Sakura), acquired in 2003, is used only by the staff of the facility. He allows the automatic dehydration and the impregnation in paraffin wax of 150 samples at the same time.

    -        1 table for inclusion (Sakura)

    The principal room is equiped for the histological studies with:

    -        2 microtomes (RM on 2145 and 2155, Leica), for realization of thin sections (4µm) from paraffin-embedded samples

    -        1 cryostat (CM350, Leica) for realization of thin sections (7µm) from frozen tissues

    -        1 Autostainer (Leica), acquired in 2003, for standard stainings (HE/HES) on frozen or embedded-paraffin tissues

    -        1 decloaking chamber (Abcys) to unmask the antigenic sites of sections and to make them available to antibodies.

    -        1 stereomicroscope, essential for training, that permits a permanent control of section quality.

    -        1 fluorescence microscope for analysis (DMR, Leica)

    -        1 Immunostaining automaton (Bond III, Leica) which allows a gain of time and a gain of quality, for both DAB and fluorescence immunostaining. The facility supplies all solutions : washing buffer, unmasking buffer, dewax solution,  kit of DAB revelation( for mouse and rabbit) or fluorescent secondary antibody (for mouse and rabbit). Users need to only bring the primary antibodies.

    -        1 slide scanner (Lamina, Perkin Elmer). This automaton is capable of scanning your whole slide in a few minutes in order to create a high-resolution virtual slide, whether it is in brightfield - histological stainings, or immunohistochemistry in DAB revelation - or in fluorescence. The virtual slides are then very easily observable with the software Pannoramic Viewer, that can be free downloaded on internet. With this software, you can zoom on the regions of interest without losing any resolution, making captures, notes, measures, manual countings. Thanks to the software INFORM, associated with the slide scanner, you can automate the analysis of your slides in term of objects counting or evaluation of marked surface.

    Our room of imaging contains:

    -        - A microscope (DMR, Leica), for brightfield and fluorescence, with the software of image acquisition (IM 1000). Three available filters : DAPI, GFP, Rhodamine. Seven objectives : x1,6; x2,5; x10; x20; x40; x63 and x100

    -        - 1 laser microdissector (Arcturus XT, Excilone) for the separation of two distinct structures initially included into the same tissue, in order to realize differential studies on the expression of proteins, DNA and RNA. This inverse microscope coupled with an infrared laser is completely motorized and piloted by the software Arcturus XT. The technique of laser microdissection allows to exclude the heterogeneousness of a tissue by isolating various cellular types or by cutting territories situated inside the same tissue. This technique comes true mainly on frozen section beforehand colored in the Cresyl Violet or immunostained (visible or fluorescence). The collection of the cellular population or the regions of interest is made by a capsule which is then going to adapt itself to a PCR tube containing the lysis buffer for the extraction of proteins, DNA or RNA.

    -        Applications: extracted proteins can be then analyzed on Western blot or on mass spectrometry on the 3P5 facility of the Cochin Institute (François Guillonneau).
    DNA can be analyzed by PCR. RNA, after amplification, can be analyzed on microarray or by RT-PCR, RT-qPCR. This work as well as the quality control can be made by the Génom' IC Facility of the Cochin Institute (Frank Letourneur).