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 我來講解一下好了： 這個問題主要是在問enzyme cytochemistry 和immunocytochemistry的差異 而就如何localiztion acid phosphatase來討論兩個不同方法 1.enzyme cytochemistry：是利用酵素的專一性和催化能力 當他和substrate作用產生product,利用trapping agent 跟產物結合 產生electron dense,藉此我們可以觀察酵素的位置 2.immunocytochemistry：是利用antibody-antigen reation 來進行標定 （location） 將我們有興趣的目標(antigene)利用免疫反應製造出antibody ,然後可以利用derict or inderict method 進行標定將我們想要 觀察的目標加上tag增加eletron dense以便觀察 -- 就是這樣大家加油呀 ^^b -- --

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