Introduction to Dynamic Histology Maggy Chabot ANAT 261-001 #260805950 Lab day : Wednesday Assignment #1 Dr. Craig Mandato McGill Faculty of Science Tuesday, October 3 rd , 2017
Section 1: Common histological methods a) H&E is the most common stain used in histology. “H” for Hematoxylin is the dye that stains basophilic structures with a purple-blue color. For exemple, keratohyaline granules of the Stratum Granulosum and most of the nucleic acids (ribosomes) have affinity to hematoxylin dye, so these appear blue or purple. “E” for Eosin colors eosinophilic structures bright pink. Usually, eosinophilic structures are composed of intracellular or extracellular proteins. Most of cytoplasm, collagen, bones and red blood cells appear pink or red. The structures do not have to be basic or acidic to be basophilic or eosinophilic. These terms are simply based on the affinity of the structure to the particular dye. (Bancroft, Layton & Suvarna, 2012, p. 173) b) The problem must have occurred at the step of removal of paraffin or rehydration of the sample. This uneven staining could be due to an improper removal of the paraffin with xylene, so there is still paraffin in the sample at the step of rehydration. This also could be due to an incomplete rehydration, which means that there is ethanol in the sample. The staining step cannot be done on a sample that still contains ethanol; it should be 100% water. (Georgia & Klatt, 1997-2017, paragr. 6) c) - Periodic acid-Schiff (PAS) is used to stain structures, which contain carbohydrate macromolecules found in connective tissues, mucus, basement membranes, etc. - Masson’s trichrome is often used to stain connective tissue. - Giemsa dye is mostly used to stain blood and bone marrow smears. (Knibbs, Paxton & Peckham, 2003)
d) Immunohistochemistry for fluorescence microscopy, a technique that identifies proteins and other molecules in cells by antigen-antibody interactions, is facilitating the diagnosis of specific tumours. The indirect or secondary immunofluorescence (IF) is a two-step incubation process. In first, a primary antibody recognizes a particular epitope on the target molecule and binds to it. After, the secondary antibody, which carries the fluorophore, binds to the primary antibody. Several secondary antibodies can bind to the same primary antibody. This will take advantage of amplification strategies by having more than one fluorophore molecules per antigen (Hoyt, Roman, Stack & Wang, 2014) . Direct labelling is often less useful because fluorescence-coupled and validated primary antibodies are expensive and you need more than in indirect IF. Also, the direct method allows less flexibility for detection techniques because of the color combinations of the target structures; individual primary antibody is needed for each color.
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