Procurement and provision of tissue samples. TPCL procures and maintains a biorepository of snap frozen and paraffin embedded tissues, provides fresh tissues for research and facilitates access to Department of Pathology archived tissue samples. Specific investigator requirements (ie, placement in particular media or sterile procurement) typically can be met, but require approval of a TPCL Co-director. In many cases, malignant and matched normal tissue samples are available. TPCL provides slides (stained and unstained) from the Shared Resource paraffin blocks, at a fraction of the cost of purchasing the whole paraffin sample. Tissues are distributed only to IRB-approved UCLA researchers. Certain requests require approval of the TPCL Tissue Advisory Board. Turnaround time for distribution of snap frozen/paraffin samples from the TPCL biorepository is 1-2 days. We require 1-2 weeks to retrieve materials from Pathology Department archives.
Pathologic diagnosis is confirmed on all frozen and paraffin-embedded samples prior to release by one of three staff Board Certified pathologists (Drs. Dawson, Dry and Said). Depending on IRB approval, it may be possible to provide investigators with additional pathology information (ie, such as node or margin status) from the original tumor. TPCL regularly monitors the RNA quality of its samples, which, overall, is excellent. Results indicate that RNA from TPCL tissues is of more than sufficient quantity and quality for microarray analysis
Histology-related services include fixation, processing, embedding and sectioning of human and animal tissues, cryostat sections (frozen tissues), sectioning for RT-PCR or other molecular studies, H&E staining and decalcification of tissues or blocks . Special stains, as performed in our diagnostic histology laboratory, are provided as well. TPCL staff can advise researchers on tissue preparation, preservation and handling, to ensure the highest quality results. Years of experience handling samples from small animals ensure that tissues are properly embedded and precisely sectioned as requested.
Laser-capture microdissection (LCM) services allow researchers to analyze specific cells within a larger sample. TPCL houses a Arcturus PixCell IIe with single-cell and fluorescence capabilities. Dr. Lee Goodglick, Ph. D., directs the LCM services for the core. TPCL teaches researchers how to perform LCM, and offers free advice on further molecular analysis (i.e., DNA/RNA extraction) of LCM samples and LCM-related research design. The goal is to make researchers self-reliant in using this technology.
Veterinary Pathology - Dr. Nora Rozengurt, DVM, PhD, is a Veterinary Pathologist who acquired her expertise in natural and experimentally induced mouse pathology over 14 years as the Director of the Pathology Section of the Laboratory Animal Diagnostic (Royal Veterinary College, London England). She has extensive experience in all analytical approaches required for these studies (e.g., gross anatomic examination, cytologic and microscopic evaluation, immunohistochemistry, ultrastructural and molecular approaches).
IHC/ISH services are directed by TPCL Co-director Dr. Said, an internationally recognized expert in IHC/ISH. Services include immunostaining of established antibodies as well as optimization of new antibodies; TUNEL assay for apoptosis is also performed. We have a large library of established antibodies available for use on human tissues (view). TPCL IHC staff have extensive experience working with both human and animal tissues. Several chromogens (eg, DAB, Fast Red) and counterstains (eg, hematoxylin, methyl-green) are available. The detection system most frequently used is the HP system using DAB with hematoxylin counterstain.
For new antibody optimization, we ask investigators to provide: 1. published papers (when available) describing the use, sensitivity and specificity of the antigen; 2.positive control tissue (or a list of appropriate positive control tissues); and 3. the primary antibody. TPCL IHC staff will determine the appropriate antigen retrieval conditions, based on any exisiting published literature and trials with negative and positive control tissues. For negative slides, the primary antibody is substituted with host species IgG. Optimization of antibodies cannot be performed as a rush order.
Digital imaging and image analysis services include state-of-the-art virtual microscopy (VM) and digital pathology (DP) (image analysis) services to the UCLA community. In VM, whole glass slides are converted to high resolution digital images (either brightfield or fluorescence) for easy archiving and retrieval, detailed magnification up to 40X, remote viewing via a web-based interface and preparation for publications and teaching – most of which can be performed from a single computer, using free software. DP includes performance of (or instruction in performing) quantitative digital image analysis studies. This includes both quantitative immunohistochemistry and analysis of other cellular characteristics (e.g., cell size). TPCL provides assistance with image acquisition and analysis, tips on sample preparation, and training on our Olympus microscope/Metamorph and Ariol systems.
Why use digital imaging/image analysis? VM/DP permits real-time discussions of histology images posted on secure websites, eliminating barriers to the exchange of pathology information with national/international collaborators/consultants, helping to reduce inter/intraobserver variability that adversely affects quantitative histopathological analysis, improving the overall efficiency of our researchers and clinicians, and eliminating delay, damage, or loss of often irreplaceable slides during shipping between UCLA faculty and extramural sites. Images can be archived, stored indefinitely, and easily retrieved - unlike glass slides, which may be lost, misfiled, damaged or fade with time.
TPCL houses several different scanners and image analysis programs (discussed in detail below): the Ariol SL-50 high throughput scanning system (fluorescence, brightfield, image analysis), Aperio XT high throughput scanning system (brightfield, web enabled) and Metamorph Image Analysis System.
APERIO WHOLESLIDE SCANNER
With the Aperio XT system, we perform wholeslide scanning. Scans can be made at 20x (default) or 40x (please, specify on your request). Images are saved to a local imaging server with 1.5 terabytes of storage in a RAID array. Users are able to view and annotate images using their computers and the ImageScope viewer software (software is both Macintosh and PC compatible). Images are delivered via CD/DVD or external hardrives along with a copy of the ImageScope software. They will be hosted for 60 days on our Imaging server; if you want us to host them for a longer time, please contact Dr Dry. The Aperio scanner is ideal for brightfield wholeslide scanning for research (H&E or IHC), education and archiving. Additionally, image files are easily converted to extensions that allow image analysis by the MetaMorph system.
ARIOL WHOLESLIDE SCANNER
Ariol is an automated scanning microscope and image analysis system. With Ariol SL 50, we are able to scan both brightfield and fluorescent slides. The software allows the linking of multiple slides so that the researcher can follow same region from slide to slide. Ariol can quickly test for multiple proteins simultaneously (up to 4 probes/fluorochromes). For the IHC slides, Ariol can perform automatic quantification of protein levels by measuring staining intensity and the pattern and location of staining in each cell (nuclear, cytoplasmic or membranar)
METAMORPH SYSTEM
Metamorph offers a complere set of processing tools ranging from background subtraction and shading correction, to morphology filters. The software can analyze a wide range of parameters, from simple intensity logging to advanced morphometry analysis, and assay-specific analysis like counting nuclei. Images can be acquired with the Metamorph system as well, the system including a high-resolution digital camera, a microscope Olympus BX50, and an automated Prior stage.
Assisted sessions: the user will bring prepared samples and a staff member will operate the system.
Unassisted sessions: can be booked only by users that have undergone the training. If users need help, we try to answer questions right away. For questions regarding advanced image analysis, please plan ahead and set up an appointment.
The download of any programs/files from the PCs is strictly forbidden. Any instrument malfunction should be reported immediately.
Training: the staff demonstrates the microscope and basics of the software. Duration = at least 2 hours. Follow-up sessions are encouraged, especially for new users and for people who want to get instruction on topics not covered during the basic session.
One PC workstation is available for processing and exporting acquired images. Available software includes: Metamorph, and Adobe Photoshop.
RULES FOR THE SLIDES TO BE SCANNED (ALL SYSTEMS):
Best results are obtained with glass slides with tissues of regular thickness (4 microns), and coverslipped. Slides with marks (such as ink dots), extra mounting medium, non-glass slides, plastic cover slips, thick labels and labels that are going around the slide present challenges for proper imaging, and may need to be removed in order to obtain an optimal scan.
Images are individually analyzed once the scanning is completed. Scans with a poor quality will be rescanned manually to achieve the maximum quality possible. Lower quality scans can be caused by faint stains, air bubbles, markings on the glass surface, making some slides unscanable. In those cases, we recommend sample recut or restaining.
Recommended software for your computer:
ImageJ = free software for image analysis (Java based).
Follow the links to download documentation, plugins.
Recommended Imaging books:
Digital Image Processing: A Practical Primer by Gregory A. Baxes
Digital Image Processing by Kenneth R. Castleman
Digital Image Processing (2nd Ed) by Rafael C. Gonzalez and Richard E. Woods
The Image Processing Handbook (3rd Ed)by John C. Russ
Video Microscopy: The Fundamentals by Shinya Inoué and Kenneth R. Spring
Video Microscopy by Greenfield Sluder and David E. Wolf
