A "DIRECT-COLORING" THIOCHOLINE METHOD FOR CHOLINESTERASES

219 Ins thue exheussivelv ussed Koelle ;onsd Fniederuwald histociueuusbcal nuetisod Ion cisolinsestersose, amid its several ns(odificat ions (see Pearse, Histocheun istry, 2moded., p. 478, Little, Brown & Co., Bostons, 1960), thse tiuiociso)biose ester used as substrate is hydroiysed by cholimsestenosse, oond the liberated hhiocisohinue is captured h)\ Cu icns, pnecipitatirug sos colorless copper thiocisolbuse. The latter is convented to broot-nisis CuS by treatiuseust ot-iths yellow aissosuonununsu sulfide. Voonious steps irs thse nusethod hsovebee-mu criticized (Ion discussion see Pearse, ibid.). Lsorge useedle-like (‘rystals (occur l)articUbsonly sot sites oof isigh activity, at higher pH levels, oomsd wihis cvenlcomsg incusbatioru. Under somuse cmcuuuushsonuces, the crystals coons soppanently increase ins sizeby nsonu -euszvussat besoccnetions . (Mabnsgren sonod Syivo mu, J. Histocheun., 3: 441, 1955). Tise so(lvsonstages ccl thie presemutly described nssethsood sore: colour is prooduced directly at the site of eoozyusuatic activity, facilitating estinsation of h.hue ooptinuuius imscusi)soti000s tinse; the l)necipitate is finselv grssusuulan, so) lhooot, eveos witis prolonged incusbosoh booms, needle-like deposits are not ob)served; instemuse cnol(on sond contnsost coon conseqtiently be l)rodltlce-ib, somuch sites 0)1 low activity ussoone easily detected. TIse bsosis ol hbue issethod is that hisiochcdimseis l)elieved Its recluse-c ferricysonside to lerrocyoonide prefenenstisolly. The lootter coomiuhimues with Cu#{176} iomus to lonuss the iossolusb)le copper femnocyoonide (Hsotcheht’s l3rcowns). Tise Cti ions in the ussedium sore cocosuplexetl wit Is cit nsote too prevent fonisuation oof copper fennicysonside. Fcon best restults I huenose-I hucod is penlconsused as fooiioows: boloocks cof tissue sore fixed overmuiglut in coold 10#{176} Ionossooiims coomsh.ooirsbrsg 1% CaCI2 aoud are tbue-ms kept ins 0.88 11 sucrose-1’ guns arabic (Holt et al., .1. Biophys. Biocheno. (‘ytol., 7, 383, 1960). TIne blocks are wasised for five nusinutes in distilled ot-sote-r, blotted dry, sonuch sore quick-frozen in iso-