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Observations are performed on a transmission electron microscope JEOL 100CXII at the LISA microscopy facility. Grids for transmission electron microscopy After staining with uranyle acetate/lead citrate, the grids are ready for observation.ĮPON resin blocks containing different fabrics appearing in black Prior to the resin embedding process, the samples are exposed to a second fixative: Osmium tetroxyde.Ģµm-sections are then stained with a mixture of Azure II/methylene blue allowing the selection of a region of interest to complete ultra-thin sections.ģ or 4 sections from 60 to 150 nm thick are set on a copper grid. The tissue sample size MUST BE less than 1mm x 1mm x 1mm. Chemical fixation and EPON Resin embeddingĪs the chemichal fixation is performed with glutaraldehyde. The facility achieve also all operations to obtain grids for Transmission Electron Microscope (TEM) May-Grünwald Giemsa: nuclei are visible in purple/blue, cytoplasm in pink, acidic granulations in dark blue, basic granulation in orange, neutral granulations in lilac/purple. Periodic Acid Schiff (PAS): nuclei are visible in purple/blue, polysaccharides, glycogen, and mucins in purple. Sirius Red: cytoplasm is yellow, collagen fibers appear in red Mayer hemalun / eosin: the nuclei are visible in purple and the cytoplasm in pink The staining is done with a ThermoScientific Gemini AS machine. The IMRB imaging facility also provides a Leica CM3050S cryo-microtome allowing frozen sectioning at -40☌ and from 5 to 100µm thichkness.įrom left to right: Microtome Finesse and cold plate, cryo-microtome. There is also a freezing plate Histostar to cool down the paraffin embedded samples and a heating plate to smooth the section. They can be can be recovered on Super-Frost+ microscope slides (for immuno-chemistry stainings), or on untreated microscope slides (for ulterior histological stainings).
SHANDON HISTOLOGY PROCESS 3 MANUAL MANUAL
Then the samples are embedded in paraffin on a Thermo Scientific HistoStar station.įrom left to right: impregnating PLC Logos, paraffin fountain HistoStar, microtome Shandon Finesseģ to 30µm sections are obtained with a manual microtome Shandon Finesse 325.
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These steps are performed by a Milestone Logos automaton to obtain the best impregnation. The preparation begins with the dehydration and paraffin infiltration of the samples. The sample size must not exceed 4mm in thickness by 2cm in width by 2,5cm in length. The tissue sample fixation must be done with formol. The IMRB imaging facility provides either an all-inclusive service (from the sample preparation to the sectioning) or a do it yourself service (after a training provided by the IMRB imaging facility staff). The IMRB imaging facility provides two services:ġ) Sample preparation 2) Optic microscopy The facility provides all the equipment needed from the sample preparation for histology to observation in microscopy. This will facilitate industrial partnerships. This certification guarantees a set of requirements concerning the quality management in the facility.
SHANDON HISTOLOGY PROCESS 3 MANUAL ISO
The IMRB imaging facility have been certified ISO 9001 since April 2014.
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The IMRB imaging facility provides expert guidance and support to IMRB staff and to external users for histology and microscopy, from the sample preparation to the image acquisition.