Research & Development
Research & Development
R&D: General Information
The RHEM platform does not only work on researcher-led projects; it also conducts technological development for the benefit of the scientific community.
R&D: Histological Phenotyping
🔬 A Unique Technology for Tissue Analysis
Unlike other technologies such as next-generation sequencing or flow cytometry, multiplex immunofluorescence (IHC) can simultaneously detect multiple biomarkers (detecting >2–6 targets, e.g., 5 + DNA) on a single tissue section while preserving tissue architecture and cellular interactions.
This technology provides spatial information on protein expression and cellular interactions within tissues, which is crucial for assessing disease progression or characterizing, for example, the tumor microenvironment.
🧭 Advantages and Innovations of Fluorescent Multiplexing
This technology is particularly valuable because certain cell subtypes require the combination of 2 or 3 markers for accurate identification. Using innovative systems such as tyramide-based fluorescent detection, sequential fluorescent multiplexing can be applied with antibodies from the same species or requiring different antigen retrieval conditions (see figure).
This approach opens new possibilities for maximizing the use of precious tissue samples and overcomes some limitations of traditional multiplexing, which is restricted to 3 markers. While less comprehensive than newer technologies like mass imaging or CODEX, it remains more accessible in terms of cost.
🧩 Scientific Objectives for RHEM
The goal for RHEM is to provide researchers with new validated antibody panels to characterize cell populations and interactions, to study various biological processes (proliferation, cell death, DNA damage, etc.), and to explore cellular signaling pathways.
R&D : Histological phenotyping RNAscope
Hybridation In Situ par la Technologie RNAscope
RHEM_AdV provides access to fully equipped workstations featuring RNAscope® technology, offers custom services and supports user in developing customised protocols tailored to their specific research needs.
RNAscope® technology enables the visualization, localization and spatially quantification of target RNA or microRNA expression at the cellular or tissue level. Detection can be performed using chromogenic or fluorescent staining, depending on the desired readout.
This technology can also be combined with immunofluorescence, allowing simultaneous visualization of RNA and protein expression within the same sample.
RNAscope
Advantages of this technology:
- High specificity and sensitivity, thanks to a double ‘Z’ probe design that minimize non-specific signals and enables strong signal amplification,
- Targeting of short regions (40-50 bases), enabling the detection of partially degraded RNAs,
- Multiplexing capability, allowing the detection of up to 3 simultaneous targets, together with nuclear conterstaining (DAPI),
- Compatibility with various tissue preparation, including microtomy, cryotomy, vibratomy.
Specific approaches available on the platform:
- RNAscope® Multiplex Fluorescent V2 with or without protease
- miRNAscope
- RNAscopePlus smRNA_RNA
- RNAscope duplex
R&D: Transparisation
Transparisation
Several chemical tissue clearing techniques have recently emerged that make animal or human tissues optically transparent. These techniques are applied to whole organs or thick tissues by homogenizing the refractive indices within the tissue. Cleared organs and tissues can thus be observed in 3D up to 8 mm deep at high resolution, without the long and tedious steps required for serial sectioning. Tissue clearing techniques are therefore highly valuable for analyzing biological processes in normal or pathological organs, both in basic biology and medical sciences.
Technology
With support from the Occitanie region, the European Union (FEDER), and the University of Montpellier, RHEM has acquired an X-Clarity tissue clearing system (Logos BioSystems), which enables highly efficient and reproducible tissue delipidation without using highly toxic solvents or reagents. For example, this system can clear a brain in 5 hours instead of 5 to 7 days manually.
The system’s Active Clarity Technique (ACT) preserves fluorescent tags from genetic modifications (e.g., YGF, GFP, Tomato, etc.), is compatible with fluorescent transfection markers (e.g., Vybrant+) and intercalating agents (e.g., DAPI, To-Pro-3), and maintains tissue antigenicity (e.g., Olig1, GFAP, NF200KD, myosin 7A, etc.). Furthermore, tissue clearing enables the use of second- and third-harmonic generation techniques (SHG, THG) for visualizing structures such as collagen and elastin fibers. It also allows the use of autofluorescence to observe the complete tissue architecture (e.g., meninges, muscle fibers, blood vessels, etc.).
According to Lee et al. (2016) Scientific Reports 6, Article number: 18631 (2016)
RHEM offers support for 3D study projects using the X-Clarity automated system. This includes tissue clearing, optional decalcification, and—depending on user requests—immunofluorescence processing, microscopic observation, and 3D reconstruction (e.g. with Imaris) in partnership with the MRI imaging platform.
Contacts
Please contact Chantal Ripoll, who oversees this technology within RHEM-AdV, via our RHEM-AdV contact form.
Mouse muscle: synaptic vesicles SV2 (magenta) and 200 kDa neurofilaments (red). Autofluorescence of muscle fibers (green) and collagen fibers in SHG (white). Collaboration N. Tricaud, INM. Leica SP8 DIVE.
Mouse paw, Nestin-GFP transgenic (green). Collagen in SHG (red). On the right, surface-rendering 3D reconstruction, Imaris, Collaboration J-M. Brondello, IRMB. Zeiss LSM 7 MRI-INM.
Spinal cord of dtTomato transgenic mouse expressing protein of interest (red). Nuclei visualized in blue (DAPI). Collaboration J-Ph. Hugnot, INM, Zeiss LSM7 MRI-INM.
Left: guinea pig cochlea after ototoxic treatment: engrafted auditory progenitor cells (Vybrant+ cells, red). Nuclei in blue (DAPI) and tissue/blood vessel autofluorescence in green. Collaboration A. Zine, Univ. Montpellier & LNIA, Marseille. Macroscope II Lavision Biotec.-Inmagic-Inmed.
Right: mouse cochlea. Myosin VIIA immunostaining: sensory cells of the organ of Corti in red, nuclei in blue (DAPI). Autofluorescence of cochlear tissue and blood vessels in green. Collaboration F. Venail, INM & CHU. Zeiss LSM 7- MRI-INM.
R&D: Biological Tissue Collection
🎯 Objective of the BTC Portal
RHEM has developed, following the model of a human biobank, a web portal for querying a paraffin block repository derived from animal models of human diseases generated by researchers in Montpellier. This portal, named “Biological Tissue Collection” (BTC), aims to contribute to the 3Rs principle (Replace, Reduce, Refine) and to promote the valorisation of valuable biological resources.
🧩 Project Content and Support
Initially, only wild-type animal models, animals developing spontaneous pathologies, or genetically modified models will be included in this BTC. The integration of animal models receiving human/murine cell grafts or human tissues will be carried out at a later stage. This project is supported by the Occitanie Region, the European Union, IBiSA, BioCampus Montpellier, and the SiRIC Montpellier Cancer.
💻 Operation and Management
The BTC portal will rely on the histology platform management software (100lims4histo-RHEM) developed by ASA (Advanced Solutions Accelerator). This software records researchers’ orders, summarizing the collected samples along with associated data about the animal and the sample – species, genetic background, genotype, age, sex, organ, etc. – as well as all technical procedures performed: embedding the sample in a paraffin block, obtaining stained or immunolabeled slides from this block, scanning the slides, etc.
ASA (Advanced Solutions Accelerator)
🌱 Valorization and Ethical Impact
Through this portal, RHEM will provide researchers the opportunity to better valorize their generated paraffin blocks by making them searchable and visible. This will increase the scientific potential of the blocks produced by researchers, giving them a “second life.” From an ethical perspective, this tool aims to reduce the number of animals used in experimental research.
