Tissue Collection:
-
- Transcardial Perfusion: This method involves perfusing the animal with paraformaldehyde through the heart to preserve the tissues and especially the brain. This ensures that the tissue samples are well-preserved and suitable for detailed analysis.
- Vibratome Sectioning: After perfusion, brains are sectioned using a vibratome. This device allows to cut very thin, precise slices of tissue, which are essential for our molecular studies. Then they are conserved in -20°C in cryoprotectant before analysis.
Molecular Techniques:
-
-
- Immunohistochemistry (IHC): This technique involves using antibodies to detect specific proteins within the tissue sections. By applying fluorescent or chromogenic labels, we can visualize the distribution and abundance of these proteins under a microscope, providing insights into the molecular changes associated with epilepsy.
- In Situ Hybridization (ISH): ISH is used to detect specific nucleic acid sequences within the tissue sections. This method allows to localize the expression of particular genes at the cellular level.
- Quantitative Polymerase Chain Reaction (qPCR): qPCR is a powerful technique for quantifying the expression levels of specific genes. By measuring the amount of target DNA or RNA in our samples, we can assess how gene expression changes in response to epileptic conditions or treatments.
-
These techniques enable us to study the molecular and genetic underpinnings of epilepsy in detail, providing valuable insights that can lead to the development of new therapeutic strategies.