1. Acid phosphatase is glycoprotein. Cytochemical location of this enzyme could be approached by traditional enzyme cytochemical method s and immunocytochemical method. If you are a professor, you are going to submit a proposal to financial-support organization. How cam you do in systematic approaches? First, what are Enzyme cytochemistry and immunochemistry Ａ．Enzyme cytochemistry utilize the function properties of enzyme to localize the site of their activity. It has played a major role in helping us understand the structure and function of cells. The general scheme of enzyme cytochemical reaction: Exogenous Insoluble Electron dense Substrate → Reaction → Product ↑ Product ↑ ↑ Enzyme trapping (precipitated) (recycled) agent (comsumed) Experiment: Using the enzyme cytochemistry method to localize the acid phospatse in guinea pig Leydig cells. 1.Fixation: 2.5hour with glutaraldehyde(1.4%) which was purposely kept very low to not destroy enzyme activity. 2.Incubation: Section was incubated by an incubation medium which consisted of the substrate, sodium β-glycerol phosphate in tris-maleate buffer, and trpping agent, lead nitrate. 3.Control: a. deletion of the substrate from incubation medium b. inhibition of enzyme activity 4.Postfixation and Tissue Processing Using the Osmium tetroxide as a postfixative 5.result Acid phosphatase activity of Leydig cells was seen in the inner cisternae of the Golgi apparatus, within lipofuscin pigment granules and autophagic vacuoles. Ｂ．The process for detecting antigens using antibodies directed specifically against them is called immunochemistry. When the electron microscope is used to detect the localization, the technique is usually reffered to as immunocytochemistry. The general approach to immunocytochemistry is to attach electron or light microscopocallt detectable tags to antibody molecules. And the tagged antibodies are then exposed to the tissue antigen of interest to which they specifically attach. Tag: 1.organic molecules that possess structured electron opacity. 2.enzyme 3.heavy metal Antibody labeling 1. Direct method ∥ ↙tag ∥ ▲ + ／＼● → ▲／＼● Tissue Primary Antigen antibody 2.Indirect method ∥ ∥ ／＼● ∥ ∥ ∥ ∥ ▲ + ／＼ → ▲／＼ + ／＼● → ▲／＼ Antigen Primary Secondary Antibody Antibody +tag Experiment: 1.Obtaining and applying the primary antibody 2.Obtaining the secondary antibody 3.Tissue fixation 4.Preembedding or postembedding labeling 5.Embedding 6.Blocking 7.Stain 8.Control a. adsorption b. use of tag or unlabeled antibody c. omission of primary or secondary antibody d. use of pre-immune sera 我来讲解一下好了： 这个问题主要是在问enzyme cytochemistry 和immunocytochemistry的差异 而就如何localiztion acid phosphatase来讨论两个不同方法 1.enzyme cytochemistry：是利用酵素的专一性和催化能力 当他和substrate作用产生product,利用trapping agent 跟产物结合 产生electron dense,藉此我们可以观察酵素的位置 2.immunocytochemistry：是利用antibody-antigen reation 来进行标定 （location） 将我们有兴趣的目标(antigene)利用免疫反应制造出antibody ,然後可以利用derict or inderict method 进行标定将我们想要 观察的目标加上tag增加eletron dense以便观察 -- 就是这样大家加油呀 ^^b -- --

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