By Prof. Dr. Zdeněk Lojda, Prof. Dr. Reinhart Gossrau, Prof. Dr. Theodor Heinrich Schiebler (auth.)
During contemporary years enzyme histochemical reactions have more and more been regarded as very important, the reason is that enzyme histo chemistry is now a well-established hyperlink among morphology and bio chemistry. the improvement of various new equipment and particularly the development of present suggestions contributed to the growth of enzyme histochemical reactions. at the present time, using those tools permits specific perception into molecular techniques of unmarried cells and their parts. the choice of an appropriate procedure for enzyme histochemical investigations wishes thorough wisdom and significant review of the reactions de scribed for the histochemical demonstration of enzymes and brought in laboratory perform. usually, it truly is tough for scientists basically curious about the applying of equipment and for laboratory assistants to touch upon the worth of an enzyme histochemical response. Our booklet will function a advisor during this recognize. It includes crucial histochemical tools for the localization of enzymes, all of that have been checked by way of the authors themselves. those tools have been usually changed and regularly used for various diverse investigations of fit and diseased organs in uncomplicated study and in regimen practice.
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Extra resources for Enzyme Histochemistry: A Laboratory Manual
Moreover, the incubation procedure depends on whether it is carried out in aqueous, semigel or gel media, with or without semipermeable membranes. The following alternatives are possible for incubation with aqueous media: Incubate in covered plexiglass boxes or petri dishes nique) lined with moist filter paper at 37 0 (drop tech- C (moist or water vapor chambers). Place the plexiglass rods on the moist filter paper, place the microscope slides with the sections on the rods, and cover the sections completely with medium, using a dropper or pipette.
The freeze-drying apparatus (Edwards, Leybold-Heraeus) should also be prepared: Renew the phosphorus pentoxide, turn on the cooling water, check the oil level; close all valves; switch on the pump; select the cold position and turn the variable controller to maximal cooling, degas the oil (pump set at "ballast") for 20-30 min; close ballast and check the vacuum performance of the pump; clean the pack rings if necessary. The container for sections consists of a brass box, a perforated cover, and a copper wire tray (Fig.
4) for 15 min-24 h at +4 0 C. Afterwards, remove the blocks from the fixative, "dry" them by rolling in cellulose or touching with filter paper, and transfer cooled Holt's mixture, distilled water, tap water, or 5%-30% sucrose solution for 2-30 h at +4 0 C. Twelve hours' treatment with water after washing in sugar solutions allows higher sectioning temperatures and facilitates the mounting of sections, but supports the formation of freezing artifacts. Better results are usually obtained without treatment with water.