T5 Flashcards
Neuroanatomical techniques
Anatomy tells us about the anatomical structure of the brain.
- Histological Procedures: Gross examination of the brain does not allow us to study details of cells, cell structure and their connections, if we want to do so we need to selectively stain thin slices of the brain.
- Postmortem tissue: After death the soft brain tissue is destroyed by autolytic enzymes, so it is very important to preserve the brain (human, mice…) with a fixative, such as formalin. Slides or sections of the brain or spinal cord are a very valuable material to study the structure of the brain
The brain or the spinal cord is then embedded within a paraffin block that can be sliced thinly using a microtome and mounted on slides. Frozen material can also be used. Specially if we use rodent tissue.
Histological stains have been developed so that cell bodies, nerve fibres and membranes can be selectively viewed.
Microtome
To cut slices
Cryostat
To cut tissue slices, also allows researchers to slice soft tissues in very thin sections (microns).
basic stains
Contrast to reveal structures that in non-stained tissues cannot be seen
For example, to visualize the cell body or soma that make up the grey matter you can use the Nissl stain which colours the soma and can reveal neuronal structures.
White matter you can stain for myelin you can use Luxol Fast Blue
Immunohistochemistry to highlight molecular targets found in specific tissues
Common steps before staining
Perfuse the brain to preserve brain structure: the blood is drained from the brain and the animal vasculature is used to deliver a chemical fixative. Afterwards the brain is immersed in fixative to have better preservations.
Embbed the brain. In paraffin wax or resin, frozen
Sections paraffin, frozen, resin sections for electron microscopy
Proceed to the staining or electron microscopy
Types of stains (4)
Haematoxillin and Eosin: nuclei cytoplasm and extracellular proteins such as collagen (rosa)
Nissl stain; neurons (lila)
Klüver and barrera stain: Myelin bundles (azul)
Golgi stain: thick sections, neurons and blood vessels (marron)
Bielschowsky’s silver stain
Bielschowsky’s silver stain is a very useful tool to detect nerve fibers. It can be used to stain axons, neurofibrils and senile plaques in the central nervous system.
This method is easy to perform and is routinely used in the study of Alzheimer’s disease together with antibody staining.
Sciatic nerve (tinción)
Toluidine blue: It stains nucleic acids blue and polysaccharides purple.
Immunohistochemistry (IHC)
Technique that detects antigens (e.g. proteins) in cells of a tissue section by using the principle of antibodies binding specifically to antigens in tissues
“immuno“ in refers to the use of antibodies, IgG used in this technique.
“histo,“ means tissue
Immunohistochemistry and confocal imaging
Confocal microscopy is a powerful imaging technique used to take detailed, high-resolution pictures of cells and tissues.
Laser Light: A focused laser beam is used to illuminate a very tiny part of the sample. Unlike regular microscopes, which light up the entire sample, confocal microscopy focuses on just one small point at a time.
- Pinholes: There are tiny pinholes that allow only the light from the focused point to reach the detector. Light from out-of-focus areas is blocked, which improves clarity.
- Scanning: The microscope scans across the sample, moving the laser point by point and building up an image, much like drawing on a screen with a pen. It does this in a series of thin slices (called optical sections).
- 3D Images: By collecting images from different layers (optical sections), a computer can create a 3D image of the sample.
Immunohistochemistry and confocal imaging (section of rat spinal cord)
stained with:
RPCA-NF-H (green): The neurofilament NF-H antibody binds primarily to phosphorylated axonal forms of NF-H, and so stains axons coursing between the large UCHL1 positive neurons. These large cells are a-motorneurons and UCHL1 protein is a major component of the perikarya and dendrites of these cells.
ubiquitin C-terminal hydrolase 1 (red).
Immunohistochemistry and confocal imaging (applications)
histological staining is routinely used to visualize individual neurons.
characterize morphological features of individual neurons.
Immunohistochemistry: the expression of individual proteins can be used to identify the location and identity of unique cell populations in the nervous system. For example, staining of the mouse cerebellum with zebrin 2 antibodies reveals the location of specialized neurons known as Purkinje cells.
