Histology Facilities
Tissue engineering scaffolds made from natural acellular tissue and synthetic polymers pose serious problems for histological evaluation due to their sensitivity, both to temperature and to the wide range of solvents used in standard protocols. We have developed a range of methods to allow the embedding, sectioning and subsequent staining or immunolabelling of both synthetic and natural materials. We have wide ranging experience of dealing with samples from in vitro studies obtained with the cell-seeded material and conditioning them under variety of bioreological and biomechanical stimulus with application of the designed bioreactor.
Embedding and fixation
Fresh tissues can be frozen or fixed using our range of fixatives designed to retain the target antigen. Samples would then be shipped to our labs where an embedding medium will be selected from various waxes and resins to match hardness of the specimen and protect heat/solvent sensitive samples. Bone samples present particular problems due to their hardness and may require demineralisation or thick sectioning followed by grinding to allow high resolution imaging.
Sectioning
Microtome equipment and tools are available in the CABER for sectioning of embedded and fixed samples. Quality of the sections satisfied optical microscopy standards, and section can be utilised for routing histological analysis and immunochemistry.
Glass knife making device are available for the researcher and postgraduate study.
Histological Staining
Once embedded, samples can be sectioned and mounted on slides for study; any remaining blocks are returned on completion. A wide variety of histological stains are available from routine haematoxylin & eosin staining (general tissue structure) to any other stain required, with common choices including:
- Masson’s Trichome (collagen & muscle)
- van Gieson (connective tissues)
- Alcian blue (proteoglycans/mucins)
- Miller’s elastin (elastic connective fibres)
- von Kossa (calcium)
Comprehensive analyses are performed for immunolabelling of tissue constructs including both immunofluorescent and immunoperoxidase labelling. Incorporated in our research are the entire process including experimental design, selection of relevant positive and negative controls, and sourcing and titration of antibodies, where available, with appropriate antigen retrieval.
Verhoeff-Van Gieson (VVG) Staining of the healthy porcine aorta versus that of an induced aneurysm in an excised porcine aorta
Verhoeff-Van Gieson (VVG) Staining of the healthy porcine aorta versus that of an induced aneurysm in an excised porcine aorta
Fig a-c show the porcine aortic wall in its excised state; 4x, 20x and 40x respectively, revealing the elastic fibres (in black) evenly distributed. Fig d-f show the porcine aortic wall which has been treated for elastin degradation; 4x, 20x and 40x, respectively displaying disorganisation of elastin [Tierney AP, Dumont DM, Callanan A, Trahey GE, McGloughlin TM (2010) Acoustic radiation force impulse imaging on ex vivo abdominal aortic aneurysm model. Ultrasound Med Biol 36 (5):821-832]
Immunofluorescence
Single or multiple labelling of your antigen(s) of interest in frozen/fixed tissues/biomaterial samples, which may be combined with nuclear stains and DIC imaging of biomaterials if required. We would also optimise relevant antigen retrieval methods if required.
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Imaging
Imaging Standard bright field and epifluorescent microscopy of thin sections is done on a research grade Zeiss AxioObserver II for immunostained and thick sections, or Nikon microscope for histological section. Both of the microscopes are equipped with transmitted light, epifluorescent and polarized light. Digital images are captured with a digital camera. Images would be collected as high resolution .jpeg or .tiff files to meet requirements.
