Preparation of Tissue for Study Flashcards
Why is the tissue cut into slices/sections to be examined?
This is because most tissues are too thick to let light pass through so the tissue is cut into small slices which are translucent and placed on glass slides for examination. The ideal microscopic preparation is such that the tissue is cut into sections so that it has the same structural features as the body. However, due to the preparation process this is not feasible as cellular lipid can be removed and there can be some distortion of the cell structure.
What are the steps of preparation?
Fixation Dehydration Clearing Infiltration Embedding Trimming
What is Fixation?
Fixation is placing small pieces of tissue in solutions of chemicals that cross link proteins and stop degradative enzymes, which preserves the cell and tissue structure. This solution is called “fixatives”. The tissue is cut into smaller pieces before fixation to facilitate penetration. In large organs, the fixatives are introduced through blood vessels which allow for rapid fixation throughout the tissue.
In light microscopy a widely used fixative is formalin, a buffered isotonic solution of 37% formaldehyde. Both this compound and glutaraldehyde, a fixative used in electron microscopy, react with the amine group (NH2) of the proteins to prevent their degradative enzyme by common protease. This
Electron microscopy provides much greater magnification and resolution and allows for fine detail (ultra-detail) to be seen. For this reason fixation must be done very carefully to preserve the cell and tissue details. Generally in EM, the glutaraldehyde treated structure is also immersed in buffered osmium tetroxide, which will preserve and stain cellular lipids and proteins.
What is Dehydration?
The tissue is transferred in a series of increasing concentrated alcohol solutions, ending with 100%, which removes all the water
What is Clearing?
The alcohol solution from dehydration stage is then replaced with an organic solvent which is miscible with both the alcohol and embedding medium and this is called clearing as the infiltration with the reagents gives the tissue a translucent appearance.
What is Infiltration?
This is when the cleared tissue is placed in melted paraffin in an oven at 52C-60C which evaporates the clearing solvent and promotes infiltration off the tissue with the paraffin.
What is Embedding?
Embedding is the process of allowing the tissue to harden in a small container of paraffin at room temperature
What is Trimming?
The hardened material with tissue and surrounding embedding medium is trimmed and placed in a microtome for sectioning.
What size are the Paraffin section?
Paraffin sections are typically cut to 3-10 um thickness for light microscopy but need to be thinner for electron microscopy at 1um thickness. The sections are placed on a glass slide and stained for light microscopy and a metal grid for staining and examination in electron microscopy.
What is staining?
Because cells are mostly colourless, they must be stained (dyed). This is done in such a way that the cell components can not only be conspicuous but also can be distinguished between one another. The dye stains the material more or less selectively, behaving like acidic or basic compounds that form electrostatic (salt) linkages with ionisable radicals of macromolecules in the tissue.
What does the term Basophilic mean?
Cell components such as nucleic acid with a net negative charge have an affinity for basic dyes and are termed basophilic. Hematoxylin behaves as a basic dye
What does the term Acidophilic mean?
Term used for the cationic components of that cell that stain readily with acidic dyes. Acid dyes (eosin, orange G, Acid Fuchsin) stain the acidophilic components of the cell such as the mitochondria, collagen and the secretory granules.
What are the most common combination of dyes used?
Hematoxylin and Eosin (H&E) are the most common combination of dyes used. Hematoxylic stains the DNA in the nucleus, RNA rich portions of the cytoplasm and the matrix of cartilage, producing a dark blue or purple colour. Eosin stains other cytoplasmic structures and collagen pink.
Eosin is considered as a counter dye, which is a single dye added separately to distinguish additional features of the tissue.
What is another fixation method with freezing?
Another quick preparation method is freezing the cell with liquid nitrogen. This however, does not deactivate the degradative enzymes but just prevents them from activating. Once the cell has melted the enzymes will once again be activated.
Examples of basic dyes?
Toluidine blue
Alcian blue
Methylene blue
Hematoxylin - most frequently used
