Sample Preparation for Light Microscopy PDF

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An overview on how your prep for light microscopy...

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Sample Preparation for Light Microscopy 1. What is a fixative? Describe the functions and purposes of a fixative.  A solution used to preserve or harden fresh tissue of cell specimens for microscopic examination. Preserve tissue in a condition as closeas possible tot hat existing during life. Fixatives stabilise cellular organisation and prevents tissue from rotting /autoysis and kills microorganisms. 2. List two types of fixative you are aware of and describe why they are different?  Formaldehyde= used routinely in histology. It is usually used as a 10% neutral buffered formalin (NBF), that is approx. 3.7%-4.0% formaldehyde in phosphate-buffered saline. Particularly good for immunohistochemistry techniques  Gluataraldehyde= glutaraldehyde is a larger molecule, and so its rate of diffusion across membranes is slower than formaldehyde. Consequently, glutaraldehyde fixation on thicker tissue samples may be hampered. It is not ideal for immunohistochemistry staining. Well suited for electron microscopy.

3. What is a buffer? Describe the functions and purposes of a buffer.  A buffer is a mixture of molecules that release or bind H+ in order to maintain a relatively stable pH. Note that the function of a buffer is NOT to keep a solution neutral (at pH 7); its function is to minimize the change in pH when base or acid is added to the solution.

4. Briefly describe the key steps in the general workflow of sample preparation for Light Microscopy  Choose sample Dissect into appropriate sized pieces  Ubiased sampling of tissue, labelled Fixation Grossing Dehydration + Clearing Embedding  Sectioning Staining.

5. Write short notes on the stages in tissue processing  Done in microscopy assignment.

6. What is the process of “Sectioning” and what instrument is used for this? Is there more than one version of this instrument? If so what are the differences between them and in what circumstances would you use one rather than the other?  Once the tissues have been embedded, they must be cut into sections that can be placed on a slide. This is done with a microtome.  Yes, different types of microtomes. Rotary microtome, ability to cut 2-3mm sections. Cryomicrotome, for the cutting of frozen samples. However, the sample temperature and the knife temperature must be controlled in order to optimise the resultant sample thickness. Ultramicrotome, preparation of extremely thin sections that are important in TEM and serial block -face scanning electron microscope. Diamond knives are used preferably.

7. What is routine histology?  In the histopathology laboratory, the term “routine staining” refers to the hematoxylin and eosin stain (H&E) that is used “routinely” with all tissue specimens to reveal the underlying tissue structures and conditions.

8. Describe the most commonly used stain for histology? How/what do the various components of the dyes work/do?  The combination of Haematoxylin and Eosin (H&E) is routinely used in staining tissue sections for light microscopy. Haematoxylin reacts like a basic dye and stains basophilic tissue components (cell nuclei, ribosomes) a dark blue or purple colour. In contrast, eosin is an acidic dye, stains acidophilic components of tissues (cytoplasm, mitochondria) a pinkish colour.

9. List some other commonly used stains and the specific tissue components they act on?  Masson’s trichrome= Used in histological observation of collagenous CT fibres in tissues.  Periodic Acid-Schiff reaction=Used in staining structures with a high proportion of carbs, such as glycogen, glycoproteins + proteoglycans found in CT, mucus and basement membrane.  Wright-Giemsa stain= used on fixed cells of blood or bone marrow smears.  Gomori Trichrome blue=used to stain and identify muscle fibres. Used to contrast skeletal, cardiac or smooth muscle.  Sudan black= Used to stain lipid-rich structures of cells.

10. What is immunohistochemistry?  This staining technique involves the process of selectively imaging antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.

11. What is immunofluorescence or immunocytochemistry?  This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. A specific example of immunohistochemistry. Two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect). Direct IF uses a single antibody directed against the target of interest. The primary antibody is directly conjugated to a fluorophore. Indirect IF uses two antibodies. The primary antibody is unconjugated and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.

12. What are the difference between these two types of staining? In what circumstances would you use both/either of these techniques?  Immunofluorescence (IF) requires special equipment, a fluorescence microscope and a designated darkened area. IHC, on the other hand, can be performed using a standard light microscope.  Fluorochrome-based signals last only 3 to 10 days, depending on the signal intensity and storage conditions. The coloured precipitate in IHC staining, remains vibrant for years, and provides an excellent permanent record.  IF fluorochromes are light sensitive, so special care must be taken throughout the staining procedure to minimize loss of signal. The enzymes and most substrates used in IHC, on the other hand, are relatively light insensitive, and therefore the staining procedure can easily be performed on the open benchtop without any unusual precautions.  Am I doing colocalization studies? If so often IF is a better way to go as I can use a confocal and truly localize where a protein is expressed in a cell in the context of the expression of another protein. Sometimes because of amplification IHC can yield "fuzzy" reaction products making colocalization or intracellular localization somewhat more difficult.

13. Describe enzyme histochemistry and a circumstance in which you would use it?  Enzyme histochemistry serves as a link between biochemistry and morphology. It is based on metabolization of a substrate provided to a tissue enzyme in its orthotopic localization. Visualization is accomplished with an insoluble dye product. It is a sensitive dynamic technique that mirrors even early metabolic imbalance of a pathological tissue lesion, combined with the advantage of histotopographic enzyme localization.  Histochemistry procedures are based on the simple premise that tissues or cells, when placed in a solution chemically react with the solution to produce a colored insoluble end product. The amount and location of the end product can then be evaluated in the context of the cell or tissue.  Principle: –(1st reaction) =(histochemical): • enzyme + substrate → product ;(2nd reaction): demonstration of the product.  Almost all enzyme histochemical investigations are performed with frozen tissue, because most enzymes are inactivated by formalin fixation.  Primary use of enzyme histochemistry is in histopathology labs is in the diagnosis or muscle disease. Enzyme histochemistry serves to detect early metabolic changes in biopsy and autopsy tissue before manifestation on H&E staining or immunohistochemistry. As such, it constitutes a valuable complement to other special techniques, i.e. immunohistochemistry.

14. Why is it important to consider the plane of section of your tissue/sample? What observations can we make based on this? 

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Specimens are carefully orientated because this will determine the plane through which the section will be cut and ultimately may decide whether an abnormal area will be visible under the microscope. Most histological sections are two-dimensional slices, from a three-dimensional piece of tissue. Exactly what will be seen on the microscope slide depends on the plane of the section, i.e. the position of the microtome cut, in relation to the anatomical structures in the tissue. Most tissues are cut in a plane, which allows the underlying structure of that tissue to be seen and interpreted most easily. For example, sections of the intestine and skin are usually cut in transverse section, so that all layers of the tissue are visible....