The Pathology Core Laboratory at the Research Institute at Children's Hospital of Philadelphia unites several core pathology components in one facility. Path Core provides basic histopathology, research immunohistochemistry, tissue microarray, and laser capture microdissection services to researchers at Children's Hospital of Philadelphia and within the surrounding academic community. We offer a full range of histopathology services for both paraffin-embedded and frozen tissue samples including tissue processing, embedding, and cutting. We also perform most standard stains as well as immunohistochemistry, antibody workup, fluorescence, in situ hybridization and TUNEL. Tissue microarrays can be constructed and our staining services may be used on slides acquired from the arrays. Sophisticated imaging instrumentation is available for both bright field and fluorescent microscopy including whole slide scanning. We also host specialized software to analyze, manage, and store data on stained tissues and arrays.
"This is a list of antibodies that we have worked up for IHC or IF staining for previous customers. We do not necessarily have everything on this list in stock. Although we do keep a handful of our more common markers in stock most of these were supplied by researchers for their project (who are sometimes willing to share). If you see something on the list that you are interested in let us know and we can help you find out if it would also work well for your project. If your marker is not on our list we can do a workup for your antibody on control tissue and help with validation before staining your experimental samples."
Computer driven tissue microarrayer
"A Leica DM4000B upright scope paired with a Spot RT/SE Slider camera provides both brightfield and widefield fluorescence imaging capabilities. Optimized for 100micron coverslips.
• 1.25x/N.A. 0.04
• 5x/N.A. 0.15
• 10x/N.A. 0.40
• 20x/N.A. 0.70
• 40x/N.A. 0.85
• 63x/N.A. 0.90
• 60x/N.A. 1.40-0.6 Oil (by appointment only)
• 100x/N.A. 1.40-0.7 Oil (by appointment only)
• Leica A4 DAPI (Blue)
• Leica L5 FITC (Green)
• Leica TX2- TxRd (Red)
• Leica BGR Triple Cube
• Leica Y5 Cy5 (Far Red)"
The core has two Microm HM 355s microtomes.
"The Cryo-Star is an electronic, optionally motorized cryostat with retraction that incorporates a unique refrigeration system for independent specimen and knife cooling. This facilitates applications in both the histology and research labs.
Other features include:
• Automatic Cryo Approach (ACA) system for exact and safe approach
of specimen towards the knife
• Automatic cutting window (only with M and/or V version)
• Ergonomic design of the unit allows user to work in either a standing
or sitting position with padded and heated arm rests.
• Intergrated vacutome system for stretching cryo-sections when
cutting and for disposal of sections and debris when trimming (optional).
• Electronic height adjustment (motorized stage only)"
Two headed optical microscope.
Vacuum infiltration tissue processor
The PALM microbeam system
• Positioning Ablation Laser Micromanipulation (PALM)
• Non-contact laser catapulting of tissue
• Automation for efficient capture and pooling of large numbers of cells
• Zeiss (Axiovert 200M) HotLInk Inverted scope offers a flexible platform for a variety of specimen types
• Objective: 5x(non cutting), 10x, 20x, 40x, 63x (dry)
• Fluorescence Filters: FITC, TxRd, Cy5
Staining to differentiate between collagen and smooth muscle
"The Russell-Movat stain is useful when studying the heart, blood vessels, and connective tissues. Because of the striking way in which it differentiates collagen, elastica, muscle, mucin, and fibrin, the Russell-Movat stain reveals early and subtle changes in the interrelationships of many of the tissues which are not apparent with routine and other special stains."
"The purpose is to demonstrate of polysaccharides, neutral mucosubstances, and basement membranes by oxidation of certain tissue elements to aldehydes by periodic acid."
"Mast cells will stain metachromatically with toluidine blue; that is, they will stain a different color form the dye solution and the rest of the tissue. The control section should show mast cells stained red-purpose (metachromatic staining). The color shift, called metachromasia, generally is attributed to the cationic or basic dye and is somewhat dependent on pH, dye concentration, and temperature. Blue or violet dyes will show a red color shift, and red dyes will show a yellow color shift with metachromatic tissue elements."
"A Leica DM4000B upright scope paired with a Spot RT/SE Slider camera provides both brightfield and widefield fluorescence imaging capabilities. Optimized for 100micron coverslips."
Digital slide scanning is available at 20x, 40x, and oil immersion magnifications.
"Slide Scanning: Whole slides can be scanned and digitized to create "virtual slides" using the Aperio scanscope CS-O and IF scanners. The images are stored on a server making remote access possible. Slides can be viewed with any internet browser or else by using Image Scope."
Our staff is available for consultation, troubleshooting, and assistance in planning and interpreting histopathology experiments and related techniques.
Training includes, but is not necessarily limited to, instruction for instrument use (how to use LCM), techniques (training a technician how to do immunostaining), and specialized topics (how to capture and analyze an image).
"The Core Lab can perform chromogenic or fluorescent labeling immunohistochemistry (IHC) by hand or using the latest in automated staining technologies. We use a Bond-Max auto-stainer from Leica Microsystems that allows us to run large batches of slides under uniform conditions."
"RNAscope® is a novel multiplex nucleic acid in situ hybridization technology, based on ACD’s unique, patent-pending probe design and signal amplification methodology. It is the only clinically effective in situ RNA detection assay available today."
"The PALM system from Zeiss allows users to microdissect individual cells or regions of tissue from their slides for downstream molecular analysis.
Why use laser dissection?
Heterogeneous Nature of Tissue
• Tumor Cells
Heterogeneous tissue may confound molecular analysis because it is impossible to determine which cells contribute to which cellular constituent of a given lysate. LMPC gives you the ability to isolate specific cells of interest without contamination from surrounding cells.
• OCT embedded frozen tissues
• Paraffin embedded tissues
• Cytology Preparations (i.e. blood smears, etc.)
• Live Cell Cultures
Isolation of DNA
• Mutation analysis
• Whole Genome Amplification
• Chromosome Spreads
• Select individual chromosomes for sequencing of translocation break points
Isolation of RNA from cryosections
• Quantitative RT-PCR
• cDNA array hybridization
• Chromosome Spreads
Isolation of protein from specific cell populations
• Amplification is not possible with protein samples so larger samples are needed
• Highly sensitive methods of protein analysis (SELDI,MALD) are now available."
"The Pathology Core has seven microtomes and two embedding stations. One set is available for walk in customer use."
"The core also provides an antibody "workup" services to test novel or untried antibodies on your tissue of interest. We have extensive experience testing antibodies in both human and animal tissues. Contact us to arrange a consultation."
"We accept wet fixed tissue specimens as well as frozen samples. Wet samples are processed on one of two automated tissue processors. The processing protocol is adjusted to match the size of the tissue specimen submitted. For instructions on submitting tissue samples please download our tissue submition guidelines."
• Level I
• Level II
• Level III
• Special stain workup
"The pathology core can construct custom TMA's for researchers who have large collections of their tissue of interest and would like to be able to perform "high-throughput" analysis.
Why do I need a TMA?
• Basic research: Validate and verify gene expression data
• Translational research: Diagnostic markers, prognostic markers, theraputic markers, response to therapy, query signaling pathways, drug discovery, tissue is a valuable resource
What do I need to make a TMA?
• Source tissue: animal tissue, clinical archival tissue, prospectively acquired clinical tissue, associated data
• Pathologist: Confirm diagnosis, select representative areas for sampling (TMA cores are representative: 20 studies to date on a variety of tumors have shown strong correspondence in IHC results on TMAs when compared to "large" sections (n=3-4 cores), validate the constructed array, score the array.
• Antibody or in situ probe to query array: phosphorylation state specific antibodies, signaling pathways
• Data management and analysis tools
• Willingness to have many collaborators
TMA data analysis
• IHC scoring
— 0 negative
— 1 indeterminate
— 2 weakly positive
— 3 strongly positive
• Scores for each individual core in TMA are collected for every stain performed
• Large data sets
• Expression data can be analyzed by bioinformatic methods developed for cDNA microarrays
• Quantitative scoring (pixel counts/percent area stained) can also be performed as well"
Arrays that have been constructed previously can be found on the core's website.
Unstained slides can be prepared from paraffin blocks or frozen tissue.
Complex sectioning is available (this is multiple non-sequential levels such as 3 sections per slide each section 50 microns apart).
"Whole slide image analysis is available using Aperio's "Image Analysis Toolbox" We can also perform analysis on site for IF images using the HALO platform from Indica Labs."
"Adobe Photoshop Lightroom software provides a comprehensive set of digital photography tools"
"Aperature gives you everything you need to do so much more with your photos. Retouch and perfect images."
"We have a singe seat license (in lab use only) for HALO by Indica Labs. HALO is a whole slide image viewer and analysis platform. HALO adds additional features and tools for annotating images, making figures, and performing image analysis. Right now we are targeting this for users who would like to do whole slide analysis on slides scanned with our Aperio IF scanner. We have the following image analysis algorithms available so far: Area Quantification (brightfield and IF), Cytonuclear Quantification IF, and Microvessel Quantification IF."
"Image Pro provides image analysis tools such as cell counting and percent area stained measurements.'"
Volocity is a sophisiticated software for visualization, deconvolution, and quantitative analysis of 3- and 4-dimensional image data.