Jeffrey P. Chang

HISTOCHEMICAL STUDY OF ADENOSINE TRIPHOSPHATASE IN CYTOPLASM

tlsat al)l)roxuusatelv (-uf adenosine tniphosl)lsittitst . at’ti ’ity (16) s ’as retaimued ims the seetiumus, ismud tluat thue cytoplasnuu of hepatic paren(Isylusal (elis, remual Pnitusary coisvolutions, mususcle fibers aI5(l mssitochsotstlnia were stained! dlarklV and! (leanly liv iuieaius of thue \Vachstein-\-Ieisel techtsi(ltie for ademsosinue tni )luosphuatase. ‘l’lie emszytuse (iensonstrateol in these structures ‘as i(ietst-ifi((l itS t 51)( 0ifiC ademuosine tniphosphatase oms the h)asis of thue following findings: it (leh)huOsI ilsonv!ated a(ienosinue tniphuosphate, but muot a(l(Isosinse (liplsOSphuate atu(l adenosine mono-

THOROTRAST LOCALIZATION BY LIGHT AND ELECTRON MICROSCOPY

Thorotrast has received increasing attention as a source of information on the effects of radiation in experimental animals and humans. It has also provided a useful tool as an experimentally applicable electron-dense material on the one hand, and remained, under somewhat limited circumstances, as an applicable x-ray opaque material in certain clinical studies. Many of these studies have provided data useful in assembling the pattern of distribution of thorotrast. The observations reported here, limited to distribution and localization of thorotrast in the rat liver and closely related findings, are part of a broader study designed to identify early cellular response to radiation, and the part it may play in carcinogenesis. Peculiar to thorotrast is the concomitant consideration of a chemotoxic, and perhaps mechanical, effect, and its role not only in cellular responses but in distribution of the substance at all stages of its presence in the organism. Much of the background of studies in the distribution of thorotrast is found in the classical studies of the response of the reticuloendothelial system to colloidal materials. Described initially as ameboid cells by von Recklinghausen (1863) ,l their function in ingesting foreign materials was indicated by Metchnikoff (1883) .2 More definitive studies with the vital stains led to full identification and description of the cellular system to which Aschoff in 1924 applied the term “reticuloendothelial system.”3 Essentially anything introduced into the body fluids collects and is concentrated in the reticuloendothelial cells either primarily or by association with plasma components? Action of the reticuloendothelial system is influenced by a number of factors such as toxins, metabolic activity, hormones, and is subject to certain reciprocal functional relations such as observed in “loading” or “blocking” effects,5*6 in which limits of function may be approached experimentally, with variable recovery. The initial observations on the distribution of thorotrast in tissue (Kadmka,? Huguenin et U Z . , ~ Bauman & Schilling,O Harris & FriedrichslO) noted the localization of the thorium particles in the liver and other portions of the reticuloendothelial system. Responses varied with species, dosage and manner of injection. In studying the distribution of thorotrast in rabbits, Wen & Jung” localized the substance homogeneously distributed in the spleen, but not other organs, 10 minutes after intravenous injection. At 15 minutes thorotrast was observed in the venous capillaries of the liver and bone marrow, free and finely granular, while the concentration in spleen continued to increase. Thorotrast particles at 3 hours were seen in Kupffer cells. The amount increased in Kupffer cells at 6 hours and at 24 hours further increased to rounded configuration. At this time finely dispersed thorotrast granules were noted in the hepatic cells. In later periods the tendency for thorotrastcontaining cells to form masses and groups was noted. Subsequent histologic studies have confirmed the pattern of uptake in the reticuloendothelial system, and thrown additional light on the variations resulting at different dose levels. A number of investigators concluded that no significant

An ultracytochemical and ultrastructural study of epithelial cells in ductuli efferentes of Chinese hamster.

Ultrastructural and ultracytochemical features of the efferent duct of Chinese hamster have been studied. The ducts are composed of two main types of epithelial cells, ciliated and nonciliated. Distinct structural and cytochemical characteristics of these cells are apparent. Presence of fibrogranular complex which is supposedly related to basal body replication was demonstrated in ciliated cells for the first time in this tissue. Thiamine pyrophosphatase activity in Golgi apparatus, acid phosphatase activity in Golgi apparatus and lysosomes and alkaline phosphatase activity on basal plasma membranes of both ciliated and nonciliated cells have been localized. However, thiamine pyrophosphatase activity was seen only on the luminal surface, apical vacuole and apical tubular structure of nonciliated cells but not on the surface of ciliated cells. Similarly, horseradish peroxidase was absorbed only by nonciliated cells. The cytochemical and ultrastructural differences between the two types of cells indicate a functional specialization. The results indicate that the ciliated cells are concerned with the transportation of the sperms and that the nonciliated cells are concerned with the regulation of fluid composition in the duct since the latter are capable of both secretion and absorption.

AN IMPROVED OPEN-TOP CRYOSTAT

‘I’lsc moustisars ismove i nut roo!uuced a sseutcnvast sit foor muse ims penforsssimug t he fresh fnazems sect iuoms technsit tse (Ciiansg monsci loon , .1. Histochem. 8: 310, 1960). By nmuavismg time hsonscl ut-heel of time nmsicnatoumime , ustilizinug simm esb)ecimOll3 olesignsecl sectionm Pltmt famnm , asic! essu obayimsg t he col)emm-t #{176} l pnimuciple, tons experimnenutmub suscoelel of this nimmochimse ut-as cramstnuscted. The detmmils have boeenm pusblished (Chang et at., 4mer. J. (‘tin. Pathol., 35: 14-19, 1961). Because of its snusuhl size, ready portability, loutcost, high efficiency toni! ease of aloersutionm, the new cryastat is sumitabbe fan Iorepansmtions of tissuses for mautimse pathologic diagnosis (Ruussebl et a!. , Ac/a Union Internal. (‘on/re le (1ancer, 16: 351, 1960; Ibanmez et al., Lab. Invest. , 9: 98, 1960) anmd in imistochsensuical rese;orch (Chang anud Han, J. His/ac/neon., iss boress). Pathologists nond sciemstists have already exhiioited considerable interest ins this mmeutcmyostat, amid its io!e sicceptansce hsms proisupted its msumsusfacttsre b)y two) commmssmencisil fimnmms in this ccousntny. After tisorotsgh testing smmsd! cniticab evmolusmot ions of t ise experissuesm! mob nscodel cr3-as! so! aver ! he pmist \emmm mommo!a. half, the issstnusnsmesmt hsos beesu neclesigmsed (Fig. I ) monmo!isuspnovecl. Time expamssiamm vmmlve is nso o isiside t he cold cimsmnsmb)er usnit sosscl the camsdemssimsg unit is emicbasec!. This not ans1 ’ ismubonaves its toppeamarste, hots! sulsa isususnes smiosobuste safety loon time o)permu!OOr. A immare iocowenfuml co)ndiemssen (1 isp.) is ensuplayec! to hmosmcns the caodirug jor oc-css, ammcl mm thennsuoostmu! limos ioeenu imsstmmlbed too ncguubsote the temmupersotoure ims the cob! chasmuhen. The temperatune of the chnusmuioen emits he reguslatec! to as low as -35#{176}C. Fan t ise eo)smsloort , cansversienuce mosmel efficiemucv of timc oopensutoon, smns ansti-fnast hcsotismg elesmmenst is busilt iiuto the usmmdcn suunlsoce of t he to1) ledge tof the could cimmomssl)cn I a ebinuuinstote frost mmmmclceomsolenusmol ioomu tof msmooist nsne frossm t he air. The consolemusi nog msmsit is sit usmutcob fmmmthen iosock ammd t iue cobol oimanusiocr muune farwmorc! to provide the operatan ut-it is ksuce-raammu. The cobcl chmumumben is slight 13cmulmmngco! too moffounol sunsmpbe space foss sectio)msimsg mind nussmssiiouslsmtiamm ool time tissues. A cconstno)l ptunel is immstmullet!ans lap oof I hoe coild chmumuuboer, withimi easy neaoim of tue openmitan. Time caistroobs inuc-lusde a enumbocntotusmc o!imil so t hsensumasttu! selectar, switches momsoi ioibot lights. A qusick-fnc-czinsg stmoge foor fncezimug tisstnc omu the nssc-t mob oliscs is loemnuussmme-mut1 nisoousmsteo! ins the cold oismimumb)cr. Tisis stmmgc, wisich is tuctivsotecl by mi se osu rmmte Fnt-o uts di nt unit , mood) osmmmuucoo bates mis smuansy sos fooumr I issuse bist-s sot time smosmue I mmmc. Wisibe onse I issuse is boci mug set-i iooneoi at hers cmmss he pncbomoncdl,

A STAINING TECHNIQUE FOR AUTORADIOGRAPHS OF NONDIFFUSIBLE ISOTOPES

Nondeparaffinized radioactive tissue sections are stained with hematoxylin and eosin by being floated on aqueous solutions for 1 hr each. The sections are then thoroughly washed, dried and exposed to autoradiographic plates or emulsions for predetermined periods of time. When desirable, both stained and unstained adjacent tissue sections can be mounted on a single slide of autoradiographic plate for exposure. Kodak DK-19 and 30% Na2S2O3.5H2O solutions are used for subsequent developing and fixing. The finished autoradiographs show excellent resolution and cytologic detail, since the gelatin remains unstained while the tissue retains its stain. Stains other than hematoxylin and eosin can be applied to this technique, provided they withstand the developmental and fixation processes.

DEMONSTRATION OF LIPIDS IN MITOCHONDRIA AND OTHER CELLULAR ELEMENTS

of diazo couplinsg cams be mseglecteti was jtustifleti for 1 lie react iou its which msnphstlmol AS was coupling withu fast gnrmset GBC at an nlkalimse pH. Irs other hsist-ochsemusical or test tube svstemuss, thse approprint emuess of usimug a tiiazo coupling react ion to riwasure emmzvmsse react iomu velocities shuould be assessed ims I ermuss of thue valties for k , 1) and t. The (litiumsioms of P fromus the emszvmuse site has not beers considered lucre t Isis is a fummsction of k 1), t-Ise radius of t Ise site, and thue diffusiomu coIsstant of P (Holt amsd O’Sullivan, Proc. Roy. Soc. London B 148: 465, 1958) aisd will tlecnense F/I its the hsistochenuical systenu but will msot inst roduce anuy error int-o tise calculatiout of K,,,. Arsother aspect of the problem of tieniving kimsetic cotustants froums a huistochsensical systeuss, the imsfltmeusce of the rate of diffumsioms into the enzvmsse site of substrate has been tlisctmssed recently by O’Sulhivams (1. Theor. Biol. 2: 117, 1962).