Customer feedback usingEM Stain : OTE (Oolong Tea Extract).
We share with you an experience of using a non-toxic EM stain like OTE,
under the direction of Elisabeth ERRAZURIZ-CERDA from CIQLE (Lyon Est Quantitative Imaging Center).
EM stain biological samples (Hepatocytes, Thrombocytes) using an O.T.E-based solution before inclusion on cell pellets.
protocol:
- Prior preparation of OTE at 0.2% (solution previously aliquoted and frozen)
• 1% glutaraldehyde fixation in 0.1M Na cacodylate buffer, pH 7.4 2h at 4 degrees
• 3 rinses in 0.1M Na cacodylate for a few hours to 1 night at +4°C
• Post fixation: Osmium tetroxide 1%+1.5% potassium ferrocyanide in cacodylate buffer in 0.1M cacodylate buffer, pH 7.4.
• Rinses in 0.1M Na cacodylate buffer, pH 7.4
• Contrast OTE 0.2% in cacodylate 1h TA
• Rinses with distilled water.
• Dehydration: ethanol 30° (5min), 50°(5min), 70°(5min), 95°(5min), 100°(3x10min)
• Substitution: Epon resin 1v. / Absolute ethanol 1v.
• Pure Epon resin impregnation under vacuum
• Inclusion with Epon +DMP30. Polymerization at 60° for 48 hours
• Contrast of ultrafine sections 5min with lead citrate
• Rinse with waterConclusion:
this contrast EM Stain agent based on Oolong Tea Extract (O.T.E) allows a beautiful visualization of intracytoplasmic constituents and represents a good substitute for uranyl acetate.
- Our contrast experience with the use of OTE on two tissues types : Hepatocyte liver and thrombocyte tissue is very conclusive with good visualization of the different cellular compartments and organelles. OTE “safe stain” allowed us to rule out toxic contrastants such as uranyl acetate.
Results :
Photo1: Visualization of contacts between mitochondria and reticulum in a hepatocyte.
- • Gly: Glycogen grains, Mit: Mitochondria, REG: Rough endoplasmic reticulum, Lip: Lipid droplet
• Photo credit: Jennifer Rieusset- CARMEN – INSERM U1060 – Lyon and CIQLE (Lyon Est Quantitative Imaging Center)
• Equipment: JEOL EUROPE JEM 1400 transmission electron microscope, Gatan Inc. Orius600 camera - click on image to enlarge the photo.
- Photo2: Counting alpha granules in patient’s blood platelets.
• DTS: Dense tubular system, Gly: Glycogen, OCS: Open canalicular system, λ-G: Alpha granules
• Photo credit: Anaëlle De Wreede- Hospices Civils de Lyon – HCL – Hemostasis Laboratory and CIQLE (Lyon Est Quantitative Imaging Center)
• Equipment: JEOL EUROPE JEM 1400 transmission electron microscope, Gatan Inc. Orius600 camera- click on image to enlarge the photo.