Currently there are three main classes of enzymes that can be localized by electron microscopy; they are hydrolytic enzymes, oxidative enzymes, and transferase enzymes. The presence of enzymes is indirectly demonstrated by electron microscopic enzyme cytochemistry, which allows the qualitative study of cellular marker enzymes, as well as the relationship between enzyme alterations and disease in both normal and pathological states. Source of content Immunohistochemistry Experimental Techniques and Applications
Operation method
Sample Preparation
Principle
Electron microscopy enzyme cytochemical reaction is divided into two steps: ① intracellular enzymes under certain conditions with the substrate for the primary enzyme reaction (the formation of primary products); ② the application of capture agent and the primary product reaction to form an electron-dense substance. Electron microscopy enzyme cell chemical capture methods, the most commonly used for hydrolytic enzymes for the metal salt precipitation method and for redox enzymes for the osmiophilic substance formation method. The principle of metal salt precipitation is that the primary product of enzyme reaction combines with heavy metals to form insoluble electron dense material, commonly used lead, cerium and so on. The principle of osmiophile formation is that oxidizing enzymes react with diaminobenzidine (DAB) to form osmiophilic intermediates, which then combine with osmium to form a high-electron-density osmium black precipitate. Move 1. Tissue sampling 10 mm × 5 mm × 5 mm in size, fixed and cut into 40-100 μm thick slices with an oscillating slicer, or cut thick slices by hand with a razor blade. When taking leukocytes and bone marrow, the peripheral blood was separated by cell separation solution. Cultured cells were removed by rubber scraping. Free cells are centrifuged (800 r/min) for 5 min, coagulated and then cut into thick slices by razor blade and rinsed. 2. Fixation Fixation was basically the same as that of immunoelectron microscopy, divided into vascular perfusion fixation (10-15 min), immersion fixation (15-30 min), and microwave radiation fixation (5-10 s), and temperature control. 3. Replace the buffer solution by the buffer solution used to prepare the incubation solution, and replace the buffer solution 3 times, each time with an interval of 5-10 min. 4. 4. Incubate the tissue slices in freshly prepared incubation solution in a vibrating thermostatic water bath. Incubation temperature and time can be determined experimentally according to the different enzymes and tissues. 5. Rinse the thick slices with the buffer used to prepare the incubation solution, usually 3 times with an interval of 5 min each time, and then rinse them 3 times with 0.1mol/L sodium dimethylarsinate buffer. 6. post-fixation with 1% osmium tetroxide prepared with sodium dimethylarsinate for 1-2 h. Embedding should be done according to ultra-thin sectioning. 7. Embedding: Gradient dehydration, resin infiltration and embedding were carried out according to the conventional method in the technique of ultrathin sectioning. 8. Ultrathin sectioning and staining: conventional ultrathin sectioning, uranium and lead staining. 9. Control experiments Control experiments for electron microscopic cytochemical reactions are likewise essential, and the following methods are mainly used: 9.1 Removal of substrate from the incubation solution; 9.2 Addition of specific inhibitors to the incubation solution; 9.3 Inactivation of the enzyme at high temperature, generally 60 ℃ for more than 1 h can inactivate the activity of the enzyme. For more product details, please visit Aladdin Scientific website.