Histology

We offer the following routine histology services: Grossing, Sectioning, Dehydration, Paraffin Embedding & Sectioning, H&E Staining, Frozen Sectioning (including non-decalcified tissues), and Cell Pellet Embedding & Sectioning.

We use a slow decalcification process with formic acid to decalcify our samples in order to preserve the histological detail as much as possible. We also perform decalcification with EDTA for those samples, which require antigenic preservation.

Routine microtomy in paraffin is oftentimes not sufficient to produce the desired results of a sample for microscopic examination as the samples may be too hard or too fragile to section properly. Resin embedding is the main alternative for sectioning if paraffin is not suitable.

Our routine embedding process involves methyl methacrylate (MMA) resin, which is compatible with immunohistochemistry. We also process in glycolmethacrylate (GMA) resins such as Technovit [7100 AND 9100] or JB-4. Histologic sections are generally prepared at 4 to 6 µm, which are stained routine with hematoxylin and eosin (H&E) or connective tissue stains such as Elastic Van Geison, or Movat Pentachrome.

• EXAKT

  
  

  For hard to cut and large specimens, we have established techniques for using EXAKT grinding technologies. Accurate to within ± 1.5 microns, the system uses abrasive papers and films to prepare the required surface. A precision controller allows the user to set the machine to grind to predetermined levels. The sections are then polished and generally stained with toluidine blue – basic fuchsin. We also developed sectioning grinding techniques with resin removal to expand staining capabilities, including immunohistochemistry.

• CRYOJANE Tape Transfer

  We have available methods for hard material sectioning without resin embedding or decalfication processes, which both are undesirable for immunohistochemistry. The CryoJane® Tape-Transfer System achieves frozen sections of near paraffin-quality as thin as 2 microns, wrinkle-free, uncompressed, fully intact, and bonded to the microscope slide. The frozen section is captured on a cold tape window as it is being cut and is then transferred to a cold adhesive-coated slide. The adhesive on the slide is polymerized into a plastic layer, securely anchoring the still frozen section to the slide. The section on the slide is now ready for fixation, staining, and/or laser capture microdissection.

For evaluation of polymer degradation in explanted devices by analytical or visual methods involving scanning electron microscopy, the preparation must contain minimal amounts of tissue. We have established enzymatic tissue digestion methods, particularly for the examination of polymer-coated stents or fully erodible scaffolds, but can also be applied to other devices as well.

Scanning Electron Microscopy [Energy Dispersive X-ray (SEM/EDX) Spectroscopy]

Scanning Electron Microscope (SEM) is a widely used surface analytical technique, which is especially useful for the evaluation of endothelium, inflammation, and thrombus in stented surfaces. It also provides high magnification images of surface topography, (up to x20,0000) at a high resolution up to 2nm. SEM, accompanied by X-ray analysis (EDX), is considered a relatively rapid, inexpensive, and non-destructive approach to surface analysis. It is useful for evaluation of surface corrosion and/or elemental analysis.

Confocal Microscopy

Confocal Laser Scanning Microscopy (CLSM) is part of our microscopy imaging services. The CLSM is a sophisticated instrument with the ability to penetrate beneath the surface of a tissue and to acquire much higher resolution images than is possible with a conventional fluorescence microscope. The CLSM has advanced optics, which eliminates all the information outside of a narrow plane of focus while, simultaneously, the activation of the molecules only takes place in the plane of focus, yielding a well-defined image. Any bleaching of the surrounding tissue or sample is minimized. The Confocal Microscopy Unit provides basic methods for capturing and analyzing microscopic images. It is also equipped for the processing of image data files. CLSM can be used on paraffin, frozen, or for whole mount “en face” tissue examination.

Transmission Electron Microscopy

Digital image analysis services [morphometry, color threshold] are offered using standard computerized software. Board Certified Pathologists are available to score, read, analyze, and report histopathological results.

Digital image analysis services [morphometry, color threshold] are offered using standard computerized software. Board Certified Pathologists are available to score, read, analyze, and report histopathological results.

Routine

Our scientists perform routine immunohistochemistry (IHC) and immunofluorescence (IF) staining utilizing known controls on fresh frozen, paraffin and resin embedded tissue sections. To identify two proteins on the same section at the same time, we can also perform double labeling immunohistochemistry using a similar approach. Immunohistochemistry services are also provided for MMA embedded sections.

Double Label

We also perform double labeling immunohistochemistry (IHC) and immunofluorescence (IF) services to localize two proteins simultaneously on the same tissue section

Cell Proliferation: Bromodeoxyuridine

IHC methodologies can also be used to measure DNA synthesis or cell proliferation based on 5-bromo2’-deoxyuridine (BrdU) incorporated into DNA where the antigen is detected using a monoclonal antibody directed against BrdU. The binding of the antibody is achieved by denaturation of the DNA by exposure to acid or heat. We offer detection of BrdU incorporation in formalin-fixed, paraffin or resin embedded tissues, cryostat sections, MMA embedded,whole mount tissues, and cell culture.

Programmed Cell Death (Apoptosis) Identification

We offer TUNEL staining based on terminal deoxynucleotidyl transferase (TdT) for the identification of apoptosis in formalin-fixed, paraffin and resin-embedded tissues, cryostat sections, and cell cultures. The end products can be visualized using peroxidase-mediated chromogen deposition or fluorophores.

Detection of surface proteins in “en face” whole mount tissues

We offer whole mount IHC staining using either single or double immunofluorescence (IF) techniques. We have developed this technology for specific examination of surface endothelialization over coronary stents and can also develop this staining technology to address specific project needs.

Quantitative Real Time Polymerase Chain Reaction (qPCR)

Real-time PCR, or qPCR, remains one of the most sensitive and quantitative tools for gene expression used today. We routinely perform both custom and pre-optimized qPCR assays for specific applications. We manage all aspects of the project from assay design to nucleic acid isolation to data analysis.

• Specific applications are offered such as microarray validation, biomarker discovery, RNAi validation, miRNA expression, etc.
• Nucleic acid is isolated from tissue, cells, blood, etc. The quantity and quality are determined by using a NanoDrop® ND-1000 and an Agilent BioAnalyzer.
• qPCR is performed by utilizing ABI 7500 Fast Real-Time PCR System.

We offer our clients expert cell culture capabilities. We culture animal and human cells from both primary explants and cell lines. Our quantitative methodologies for protein expression include techniques such as cytokine antibody arrays, ELISA, and Western Blots. Further, we have established bioassays for assessment of cell migration, proliferation, and apoptosis. In addition, we have developed novel in-vitro models to assess design characteristics (material, coating, drug, geometry, etc.) on cell morphology and function.

• Coupons

  
  

  The coupon system provides a rapid method to assess the impact of a material and/or coatings on cellular behavior. Small metallic disk with customized with surface etchings and/or polymers of interest are seeded with cells and maintained under culture conditions. Thereafter, the cells can be analyzed for specific protein markers (ex: function, inflammation) using standard techniques or proteins of interest visualized by laser confocal microscopy.

• Bioreactor

  
  

  The bioreactor system provides a more relevant bench model as it can mimic the mechanical environment of an artery. The testing platform is an optically clear compliant silastic tube with geometry similar to a coronary artery. The tubing can be seeded with endothelial cells with constant exposure to physiological flow and pressure conditions. Clinical devices such as coronary stents can be deployed within the endothelial seeded tube to provide a model to discern discrete factors associated with the design. Evaluations include measuring changes in pressure and flow, assessment of device deployment, and assessment of cell morphology, proliferation and function.