Preservation of Tissues
We fix tissues in formalin as standard, implement this by machine and embed it with paraffin. This prevents tissue decay and makes it possible to cut the tissues into thin sections with good morphology. The paraffin blocks can be stored for a long time at room temperature.
Tissue is fixed in formalin for 24 hours and passed through overnight. The feed apparatus processes the material in a number of steps, including a post-fixation in formalin, an ascending alcohol series, which expels the water and a xylene treatment that makes the tissue susceptible to the paraffin.
Fixation with formalin causes crosslinks between biomolecules and degrades proteins and nucleic acids into small fragments. This can be disadvantageous for follow-up experiments. It is possible tissue not in formalin but e.g. to fix in ethanol and then to pass through without formalin, to prevent cross-links. There are also protocols that remove cross-links in formalin-fixed material.
Cutting a paraffin coupe without folding or tearing is a job. Analysts who are very skilled in cutting sections work in the PARTS laboratory. Their experience allows them to process paraffin blocks quickly and with high quality into coupes that can be used for many purposes.
The histology laboratory is equipped with iso-certified equipment with which not only the standard hematoxylin & eosin (H&E) coupe can be made, but also various special colorings such as elastin or fibrin. In the part Histochemistry you can find more information about the many possible colorings that PARTS offers. Extra blank sections can be cut and pasted if it is not yet known which colorations or determinations will follow. These sections have a reasonable shelf life (possibly cooled / airtight) and are suitable for immunohistochemistry, molecular pathological and / or FISH techniques.
For DNA, RNA or protein isolation, sections are collected in Eppendorf cups (link to molecular pathology). If laser microdissection is desired, the sections are glued onto membrane slides.
You can also use our paraffin microtome or cryostat (frozen sections) yourself. After an introduction by a PARTS employee you can use these devices at a fixed rate: paraffin microtome 10 euros per hour, cryostat 20 euros per hour. The proceeds are used for, among other things, disposables and the maintenance contracts on these devices.
IHC and IF are both proven methods to detect proteins in situ. At a glance it becomes clear which cells express a certain protein. IHC and IF stains are performed on freezer sections and paraffin sections. For IHC and IF use is made of the automatic color stations Ventana BenchMark Ultra and the Ventana Discovery. This platform has been developed for the fully automatic execution of IHC and IF on histological or cytological samples.
PARTS works with validated antibodies. We have experience with more than 450 research antibodies. For questions or experience with specific antibodies, you can send an email to firstname.lastname@example.org. When using non-routinely applied antibodies and antibodies that have recently been developed, these must first be validated.
Tumors are heterogeneous in their composition and contain various types of cells. It is possible to isolate individual cells from the tumor and then separate them from each other on the basis of different phenotypic markers. The different cell types can then be studied further. The sorting of cells can be done by FACS or with magnetized antibodies.
By making cytospin preparations, the cells can be used for immunocytochemistry and related fluorescence techniques.
For standard immunohistochemical techniques, it is sometimes desirable to pass the cells to paraffin. This is possible with the cellient technique, or by feeding cell pellets.Because biomolecule isolation techniques are constantly improving, it is possible to isolate enough DNA, RNA or protein from several hundred cells for "omic research"
Tissue bank and freezer compartment
For molecular research, frozen tissue is the best starting material. Tissue can be stored specifically for your research in the tissue bank. There is also the possibility of isolating DNA, RNA and proteins from this tissue.
The tissue bank, like the rest of the pathology department, operates in accordance with ISO15189: 2012 standards and strives for a high level of quality. Internal evidence-based research into the best procedures has created an evidence-based protocol that guarantees quality. A quality check is also carried out every year, the results of which can be found in the annual report. Central quality of samples also guarantees the quality of your prospective collection.
The tissue bank is also skilled in isolating biomolecules from freeze tissue. By cutting frozen sections, the morphology can be linked to what is ultimately measured in DNA, RNA or proteins. Moreover, in combination with laser microdissection, a specific part of a freezer compartment can be selected very precisely for biomolecule isolation. This makes the final results more reliable and also reproducible.
It is of course also possible to use frozen sections for immunohistochemistry and other histological stains. New antibodies in particular are not suitable for use on FFPE coupes. Because proteins in frozen sections are kept in their native state, frozen sections offer the possibility of demonstrating the proteins through IHC in this case.
Techniques and support for molecular analyzes of human or animal material operational within the laboratory:
DNA / RNA isolation
* from FFPE, freeze tissue, routine (colored) sections, blood, serum, plasma, urine and cell cultures
* manual microdissection of tissue sections
* Sanger sequencing
* Mutation specific PCR
* SNaPshot analyzes
* Fragment analyzes
* MSI analyzes
* Tissue / cell line identification (STR genotyping)
Next generation sequencing (Ion Torrent)
* Targeted analyzes with customized panels on DNA and RNA
* Bioinformatics support for generating and analyzing data
RNA expression (RNAscope)
In situ hybridization
RNAscoope is an in situ hybridization technique that detects the mRNA in tissues through the use of highly specific target probes combined with chromogenic detection. PARTS offers fully automated RNA-ISH with the RNAscope system from ACD-Bio. Ready-made probes from the catalog are available, but self-designed probes can also be used with this system.
microRNA detection in FFPE sections
microRNAs are small non-coding RNA molecules about 18-25 nucleotides in length that are involved in post-transcriptional gene regulation. microRNAs are involved in various essential biological processes, including cell development, differentiation and diseases. microRNAs are highly tissue-specific and play a key role in the differentiation and maintenance of tissue identity. The function of most miRs is still unknown. Deregulation of microRNAs has been associated with the development and progression of cancer. In addition, microRNAs have been identified in many studies as new diagnostic and prognostic biomarkers.
Recently it has been possible to locate individual microRNAs. This can be done in formalin-fixed paraffin-embedded tissue.
PARTS uses the Ventana Discovery Ultra with which the microRNA ISH is fully automated. It is important to use the correct positive and negative controls. After a titration of the specific probes, a standardized protocol is used.
A virtual microscope can scan tissue sections and digitally store these images at a high resolution.
The Pathology department has a Hamamatsu Nanozoomer 2.0 HT digital slide scanner (virtual microscope) for scientific use.
The NDP viewer software allows you to view the images in all magnifications and to export parts of them for publication. Measurements and annotations can also be made. With additional image processing software it is possible to further analyze the images.
You can also make the images available and have them assessed by others via the intranet within the Erasmus MC or via the internet. After being trained by one of our PARTS employees you can use the Nanozoomer virtual microscope yourself.
Tissue Micro Arrays
Tissue Micro Arrays
- Large numbers of tissues are used for research into disease-related genes or gene products
- High-throughput molecular analysis of tissues is possible with the tissue microarray (TMA) technology
- A TMA contains hundreds of tissue pieces from original donor paraffin blocks
- With 1 section of a TMA you can analyze all tissues simultaneously with a small amount of reagent
- The samples are on 1 slide, which means that experimental variability is smaller
- TMAs are suitable for morphological studies, histochemistry, immunohistochemistry, DNA and RNA in situ hybridization
- PARTS has a fully automatic TMA Grand Master
- After an introductory course, researchers can create their own TMAs
- PARTS employees can do this work for you
- The TMA grand Master also offers the possibility to collect the cores in eppendorf tubes for other molecular research
- You can also use our TMA Grandmaster yourself. After an introduction by a PARTS employee you can use this device at a fixed rate of 20 euros per hour. The proceeds are used for, among other things, disposables and the maintenance contract for this device.
Electron microscopy is a very powerful technique that studies the internal structure or surface morphology of materials and biological objects. Electron microscopy reveals details of a few nanometers. PARTS offers electron microscopy techniques with a focus on sample preparation and image analysis. PARTS has a Morgagni 268 D, Transmission Electron Microscope. PARTS employees can help you with the optimal experimental conditions for your project. We have experience with a wide range of biological specimens.
- Chemical fixation
- Embed plastic
- Electron microscopic imaging
- Image analysis
Laser Capture Microdissection
- Tissue heterogeneity makes it difficult to interpret data obtained from analyzes of cells with morphological and phenotypic differences. The PALM laser microdissection technique can isolate single cells or histologically pure cell populations from tissue sections, which are important for your research.
- The cells of interest can be separated from the rest by microdissection and used for DNA genotyping, RNA analysis, cDNA generation and proteomics, among others. Microdissection is possible on both FFPE and fresh frozen tissue.
- The isolation is possible from existing coupes. If the coupe still needs to be manufactured, it is best to use specially made membrane slides.
- You can use the equipment independently after an introductory course. An appointment can be made for this via: email@example.com
- The PALM can be reserved on the intranet via the Optical Imaging Center booking system under "Equipment booking". The costs are € 20 per hour for financing the maintenance of the system. You can also have the micro dissection performed by PARTS employees.
Rental laboratory equipment
You can also use the following equipment at an hourly rate:
- Hamamatsu Nanozoomer slide scanner
- TMA Grandmaster
The equipment is available after an introduction by a PARTS employee. The costs for introduction are €30. Please contact us for a quotation by sending an email to firstname.lastname@example.org