WUCCI – Frequently Asked Questions
Where do I start?
News users should peruse the New User section of the website. Much of the information you need will be there. Also, the Scientific Director is your first point of contact with regard to accessing WUCCI resources.
I would like to use a WUCCI microscope. How do I do this?
Microscopes within the WUCCI are available ONLY to those researchers who have satisfactorily completed training via an authorized WUCCI staff member. A member of your lab or another lab may NOT train you, only trainings conducted by WUCCI staff will result in you being able to sign up and use a microscope as needed. Detailed instructions on microscope use can be found on the Facilities pages.
I know there are costs to using the WUCCI, what are they?
All costs for microscope use, sample preparation and data analysis are posted on the Recharge Rates page.
At what times can I use the WUCCI microscopes?
All microscopes are housed in the WUCCI, which is a self-contained, secured space in the basement of the McKinley Research Building at 4515 McKinley Avenue. Access to the building and to the WUCCI is via a valid WUSTL ID badge.
All light microscope systems are available for use 24/7 assuming proficiency in use has been demonstrated and the Scientific Director has authorized access. The TEM and FIB-SEM are available Monday through Friday 9:00 a.m. – 5:00 p.m. with use after hours or on weekends limited to users with extensive experience who have undergone additional training.
Please remember that ALL microscope scheduling events and EM sample preparation requests should be submitted before use through the iLab Solutions on-line management system.
I need help planning my experiment. Can you help me?
For new experiments or for inexperienced researchers it is best to first consult with the WUCCI Scientific Director to insure that specimen preparation is optimized and to ensure you have a through understanding of microscope use. During that consultation, the Scientific Director can help determine feasibility, provide suggestions for optimal use of the WUCCIs resources, and, if appropriate, alert the WUCCI staff to unique details of specimen preparation for your project. Post-meeting, the Scientific Director will work to facilitate a training session and/or alert WUCCI staff to incoming sample preparation requests.
How do I prepare my samples?
This is a complex question and depends very much on the sample, the fluorophores in question, and the information being desired. Please contact the Scientific Director first to discuss your project.
This is an easier question to answer since the user is only really responsible for fixing the sample; the WUCCI EM staff undertakes the majority of post-processing and staining steps.
- Fixation. Samples must be fixed prior to submission. If your project involves immuno-labeling you MUST first discuss your project with the Scientific Director and a member of the EM WUCCI staff prior to preparing and dropping off your samples. We typically recommend fixing samples in a 2.5% glutaraldehyde + 2% paraformaldehyde solution in 0.15M sodium cacodylate buffer at 37˚C with a solution volume greater than ten times the sample volume. Please contact the WUCCI EM staff to obtain pre-filled specimen vials of fixative solution. SEM samples should also arrive at the WUCCI fixed, however the WUCCI EM staff will take care of dehydration and critical point drying prior to mounting and imaging in the SEM.
- Tips and Tricks. In order to maintain cellular ultrastructure, it is critical that specimens are fixed immediately after the oxygen supply has been terminated. The exact specifics of the fixation procedure (fixative, temperature, time and concentration) will vary on the sample / experiment. However, typically a buffered aldehyde, which serves to cross-link proteins, carbohydrates and sometimes lipids, is used. Such a fixative may also cross-link similar molecules that surround the sample (body fluids, cell culture medium, etc.) so the sample should be cleanly washed in a buffered solution before fixation.
Aldehyde fixatives penetrate tissue slowly, so it is important to ensure a small sample size (<0.5 mm on two sides). In addition, low temperatures depolymerize cytoskeletal elements, thus it is critical that buffer washes, fixatives, and samples should be kept at room temperature or warmer to initiate fixation. To store fixed samples for more than a few hours, the specimen should be placed on ice or in the refrigerator after fixation. For storage of samples more than a few days before processing, the fixative should be replaced with buffer after 24 hours.
I am ready to submit samples for EM processing, how do I do this?
Firstly, please ensure that you have discussed your project with the Scientific Director and a member of the WUCCI EM staff prior to submitting any samples. All processing requests MUST be submitted though the iLab Solutions on-line management system. Please contact a member of the WUCCI EM staff if you run into problems submitting your request.
Please note that all specimens submitted for EM processing should be fixed prior to arriving in the WUCCI and should be kept refrigerated until submitted. Please coordinate with a member of the WUCCI EM staff to obtain EM fixative and washing buffer.
I need more help? I’m confused? What do I do now?
Simple answer – Just ask! We are here to help!