Microscopy UT Southwestern Medical Center EMCF
Sample Preparation and Fixation Table of Contents Processing Ultramicrotomy Imaging IMPORTANT NOTICE
Training Request Form Project Request Form The user will have samples completed before requesting a training. The user will give a brief presentation of their project, include the exact tissue type you are using. Create one training request for each type of training. Include images of the orientation you would like your sample to be cut, and reference images of what you are looking for within the tissue. Check all boxes you require of our services. The user must have a sample manifest.
of tissue or 120-200 nm vibratome sections (preferable) • Fixative and Perfusion: 4% PFA, 2.5% Glutaraldehyde, in 0.1M Sodium Cacodylate buffer Sample Preparation/Fixation • Temperature of the fixative should be the temperature of the tissue/cells. • Fix tissue for 1 hour then refrigerate overnight • Fix for monolayer of cells at least 10 minutes at ROOM TEMPERATURE • Submit to the Core
• Washed in .1M Sodium Cacodylate Buffer (3x7min) • Post fixed with 1% Osmium Tetroxide(1hr) • H2O wash (6X2min) • Pre-stained with 0.5% UA in 25% Methanol (overnight) • H2O wash (5x7min) • Post stained in Lead Aspartate (30min) • H2O wash (3x5 min) • Dehydrated in graded washes of Ethanol (1hr 10min) • Infiltrated with ratio mixtures of Ethanol and Epoxy Resin. 2:1, 1:1, 1:2 (2hr, 2hr, overnight) • Complete infiltration and polymerization with 100% Epoxy Resin (6hrs of infiltration and 24hrs of polymerization)
• Block face is trimmed to shape the tissue using a single edged razor. • Using a glass knife the block is faced until the desired tissue is present. • Thick sections, ~350nm, are taken and put onto a slide. • The sections are stained with toluidine blue and approved/denied by the user. • Thin sections, ~78-85nm, are cut and collected on the desired grids. • *The grid, pictured right, indicates size of thin section* • Please see these Ultramicrotomy. videos • Block Trimming (link to video) • Thin Sections (link to video) below on
• Once the users project is complete, they will be contacted. • The user will then decide if they would like to be trained to image. • If they do, they will need to request a training on PPMS. • If not, we will image for the user at an increased rate. • *JEOL 1400, recognize NIH Grant 1S10OD021685-01A1 in any publications that result from the use of this instrument.*
• At the beginning of the new year, every year, an email will be sent to users whose samples we have stored in our facility • This email will instruct you to come pick up these samples and where they will be located in our facility • If they are not picked up by the date in the email they will be DISCARDED! • You will have a sufficient amount of time to pick them up, usually two weeks • If you are travelling during that time, please let us know
• “Ultramicrotomy: Trimming a Resin Block.” YouTube, uploaded by Microscopy Australia, 2010, https://www.youtube.com/watch?v=aytszxLiiAw. • “How to Cut Thin Sections Using an Ultramicrotome.” YouTube, uploaded by Denise Gautreau, 20th April 2014, https://www.youtube.com/watch?v=Nl-pE667M0s.
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