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Principle of H&E Staining Hematoxylin when oxidized form heamatin.

The product of oxidation of hematoxylin is actually the active ingredient in the staining solution. The non-oxidized hematoxylin is not a dye because chromophore is not present in its molecule. Addition of strong oxidant affects the in situ oxidation of hematoxylin which is sodium iodate. The oxidized product of hematoxylin, heamatin, displays indicator-like properties, being blue and less soluble in aqueous basic condition, and red and more soluble in alcoholic and acidic solution. In acidic conditions heamatin binds to lysine residues of nuclear histones by linkage via a aluminum. To ensure the saturation of chemical binding sites, the stain is applied longer than necessary, resulting in the over staining of the tissues with much non-specific background coloration. This undesirable coloration is selectively removed by controlled leaching in an alcoholic acidic solution, (acid alcohol), the process being termed "differentiation". Differentiation is arrested by returning to an alkaline environment, whereupon the haematin takes on a blue hue, the process of "bluing-up". The haematin demonstrates cell nuclei. Full cellular detail is obtained by counterstaining with the eosin mixture. There are three commonly used forms of eosin - eosin Yellowish (tetrabromofluorescein, disodium salt CI 45380), eosin Bluish (the dinitro- dibromo-derivative CI 45400), and eosin Alcohol Soluble (the ethyl derivative CI 45386), the former is preferred. Color enhancement is achieved by fortifying the stain with phloxine, a chemical member of the same family as eosin (halogenated fluorosceins). The mechanism of their staining is not fully understood, but is believed to be of an electrostatic nature. Visualizations most acceptable to the histologist are obtained by applying the dyes in acidic conditions, whereby more intense specific colorations are obtained, the more acidic tissue components taking up the dye to a greater intensity, hence the addition of acetic acid. References for H&E :
Mayer P,(1896),Mitt. zool. Stn. Neapel.,12,303 Lillie RD,(1965),Histopathologic Technic and Practical Histochemistry,3rd edition, McGraw-Hill Book Co., New York Lynch MJ, Raphael SS, Mellor LD, Spare PD and Inwood MJ, (1969), Medical Laboratory Technology and Clinical Pathology, 2nd edition, WB Saunders Co., Philadelphia London Toronto LG Luna, Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, third edition, McGraw Hill Reference for TUNEL assay:

Rode, H. Apoptosis, Cell Death and Cell Proliferation. Mannhelm: Roche Applied Science.

Methodology 3.1 Plant Collection and Identification Moringa oleiferas leaves will be collected from Sitio Bugtong Brgy. Pangao Lipa, Batangas. After obtaining the plant, its authenticity will be verified by the curator of the UST Herbarium. Identification manual will be used for further verification of plant authenticity. The obtained plant from Lipa Batangas will be compared against material in the herbarium, again for thorough verification of plants identity. 3.3 Acquisition of Test Organisms and Permits Sprague-Dawley rats will be obtained from either RITM, BFAD, or UP depending on its availability. The