Protocols

Specimens are fixed with an appropriate aldehyde fixative for a minimum of an hour, specimens should be placed into the fixative immediately, do not allow to air dry. Specimens and fixative should be kept at 4°C. Do not put them on ice, or allow to freeze. Ideally, specimens should be no larger than 3mm in width. Fixative routinely used by the center and provided, 3% Glutaraldehyde in 0.1M Cacodylate buffer. After primary fixation, specimens are rinsed in the appropriate buffer and post fixed in 1% Osmium tetroxide in buffer for at least 1 hour. After rinsing in buffer the specimens are dehydrated through a series of graded alcohols from 70-100%, after 2 changes with an intermediate solvent, 100% acetone, specimens are placed in a 50:50 mixture of acetone and embedding media (Embed 812, Electron Microscopy Sciences) for a minimum of overnight. The following day specimens are placed into a fresh change of pure resin for at least 4 hrs., then embedded in another fresh change of 100% resin, and placed in a 60°C oven for 12-18 hrs. for polymerization. The blocks are then ready to section.

The resin blocks are first thick sectioned at 1-2 microns with glass knives using an Ultracut UCT (Leica, Bannockburn, IL) and stained with Toluidine Blue. These sections are used as a reference to trim blocks for thin sectioning. The appropriate blocks are then thin sectioned using a diamond knife (Diatome, Electron Microscopy Sciences, Hatfield, PA) at 70-90nm (silver to pale gold using color interference) and sections are placed on either copper or nickel mesh grids. After drying on filter paper for a minimum of one hour, the sections are stained with the heavy metal Electron Microscope Sciences, UA replacement stain for contrast. After drying, the grids are viewed on a Spirit (FEI, Hillsboro, OR). Digital images are taken with an AMT CCD camera and are uploaded to a Box folder for the researcher.

Tissue samples are fixed with 2-4% Paraformaldehyde in 0.1M phosphate buffer, dehydrated through a graded series of ethyl alcohols and embedded in Unicryl (Electron Microscopy Sciences, Hatfield,PA). Thin sections (70-90nm) are mounted on Formvar/carbon coated nickel grids. After rinsing (see note above) with 0.1M Phosphate buffer or PBS, the grids are placed into the Blocking buffer for a block/permeablization step of 30-45 minutes.

The grids are then placed in the primary antibody overnight at 4°C. During the immuno-labeling process, the grids are not allowed to dry out. The grids are rinsed with phosphate buffered saline (PBS) and then floated on drops of the appropriate secondary antibody attached with 10nm gold particles (AURION, Hatfield, PA) for two hours at room temperature. After rinsing with PBS, the grids are placed in 2.5% Glutaraldehyde in 0.1M Phosphate buffer for 15 minutes. After rinsed in distilled water, the grids are allowed to dry and then are stained for contrast with uranyl acetate. The samples are viewed with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR). The block/perm buffer consists of 2% BSA, 0.1% Cold Water Fish Gelatin and 0.1% Tween in PBS. The primary and secondary antibodies are diluted in an incubation buffer containing 0.1% BSA-c (AURION), 0.05% Tween in PBS. Times and dilutions are determined for each particular primary antibody being used. Other types and sizes of gold can be used from 1.4nm-25nm. 1.4nm sized gold requires silver enhancement to visualize at the TEM level.

After fixation in 2-4% Paraformaldehyde, tissues samples are vibratomed (50 microns). After rinsed in phosphate buffered saline (PBS), the sections are placed in 0.1% sodium borohydride for 15 minutes to quench the aldehydes. After rinsing in 0.1M phosphate buffer until all the bubbles are gone (do not use PBS), the samples are placed into a Blocking buffer for the block/permeablization step for 45 minutes. The samples are then ready for incubation in the primary antibody overnight at 4°C. The sections are rinsed with PBS and placed into the secondary antibody attached to 10nm gold particles (AURION, Hatfield, PA) for two hours at room temperature. After rinsing in PBS the sections are placed in 2.5% Glutaraldehyde in 01.M phosphate buffer for 30 minutes. After rinsing in PBS the sections are post fixed with 0.5% osmium and processed for standard embedment using Embed 812 (Electron Microscopy Sciences, Hatfield, PA). The block/perm buffer consists of 2% BSA, 0.1% Cold Water Fish Gelatin and 0.1% Tween in PBS. The primary and secondary antibodies are diluted in an incubation buffer containing 0.1% BSA-c (AURION) and 0.05% Tween in PBS.

Fix the specimen with the appropriate fixative. An optimal concentration and clean specimen is needed for the best negative staining. The specimen is dropped onto a 200-400 mesh carbon/formvar coated grid and allowed to absorb to the formvar for a minimum of one minute. The excess liquid does not need to be wicked off. A drop of the negative stain is placed on the grid for the appropriate amount of time for the stain being used and type of specimen. The center uses Nanovan (Nanoprobes, Inc. Yaphank, NY). The time in the stain is very short, generally less than one minute, and this time needs to be worked out for the specimen being used. The excess liquid is then wicked off and the grids are allowed to dry. After the specimen is placed into the TEM, investigators should allow the specimen to sit for a few minutes so the sample can be vacuumed dried before being irradiated. The sample is then ready for viewing and images.

Users may become independent users of the T12 Tecnai by undergoing three training sessions with a center staff member. These sessions cost $500 each and will be conducted consecutively based on staff and user availabilities. By the end of training, users will be allowed to book time on the TEM through iLab and will be charged the unassisted user fee of $45/hr. Independent users will be required to undergo a refresher training each 12-month period. To schedule this refresher course, users will coordinate with staff and book one hour on the TEM. This refresher course will last 10-30 minutes depending on individual user needs. The one hour booking will cost the unassisted usage fee (currently $95/hr) and users may use the remainder of this hour to work on their own sample imaging.

Center staff will determine whether or not a user is ready to operate the center equipment independently regardless of prior training or experience.

To maximize the amount of serviceable user time on the instruments, center staff may perform operations for most users. Untrained users will defer to the center staff for all sample preparation and microscope operation as discussed on a project-by-project basis. All users are able to reserve time on the TEM through iLab, but only trained users will be charged the unassisted fee for usage. 

Following extensive training and approval by the center staff, some users may be allowed to perform some of these tasks independently. Adequate training to operate the TEM alone will be performed by the center staff. Once users have completed the required user training, staff will notify the center director and program manager, who will then give access to trained users to certain iLab reservation features. Trained users are able to reserve unassisted time on the TEM through iLab and will be charged the unassisted rate of $45/hr. All users, trained or untrained, are required to follow the user policies and procedures as listed in the "Training and Access Section."

To gain independent usage of the Tecnai, the user needs to enroll in the center's training program by contacting Mandy Bittner at mabitt@iu.edu. The user must demonstrate to core staff that they can operate the scope independently. A minimum of one training session is required, but additional sessions may be needed until the desired level of proficiency is achieved. Each session is $500 for 3 hours. Users are deemed independent by approval of core staff. If in the future the trained user shows they are unable to operate the scope independently, core staff will address this with the user and/or PI and schedule refresher courses.

The TEM is very complex with many potential points of failure. Unforeseen circumstances could potentially interrupt a user’s session. Center staff will do their best to minimize interruptions within their control. However, if a system failure does occur, the billed usage will be rounded to the nearest hour.

Users with an interrupted session will need to sign-up for a new session at the next available time. Please utilize iLab for this reservation or contact Mandy Bittner.