TETRAZO.LIUM FOR THE CYTOCHEMICAL LOCALIZATION OF SUCCINIC DEHYDROGENASE Cytochemical and Cytological Studies of Sarcoma 37 Ascites Tumor Cells

Succinic dehydrogcnase (SDH) was localized in the mitochondria of Sarcoma 37 ascites tumor cells by the use of tetranitro-BT (TNBT) and nitro-BT (NBT) in smear preparations. Results with each tetrazolium salt as electron acceptor wcrc evaluated with respect to: (a) size and shape of the formazan precipitate relative to standard mitochondrial morphology; (b) crystallization phenomena of reduced dye; (c) lipid adsorption of formazan. The association of formazanor iron hcmatoxylin-stained mitochondria with lipid droplets within the cells was investigated, as was also the influence of formalin fixation, with and without cold acetone prctreatment, on mitochondrial morphology and enzymatic staining. Data from these studics appear to indicate that T N B T is more suitable than NBT for use as a cytochcmical reagent in oxidative and /o r dehydrogenase enzyme histochemistry and cytochemistry. I N T R O D U C T I O N In 1956, Tsou et al. (21) reported the synthesis and cytochemical evaluation of several tetrazolium salts for use in histochemistry as indicators of certain types of oxidative enzymatic activity. As a result of these studies, 2,2P-di-p-nitrophenyl 5 ,5 t diphenyl 3,3 p (3,3 ~ dimethoxy ° 4 ,4 tbiphenylene)-ditetrazolium chloride (nitro-BT or NBT) was prepared, and this has since played a prominent role in the localization of enzymes such as succinic dehydrogenase (SDH), D P N H and TPNH-diaphorases or tetrazolium reductases (5-7). In addition to NBT, 2 ,2 ' ,5 ,5 ' te t ra-pnitrophenyl 3 ,3 p (3,3 p dimethoxy 4 ,4 tbiphenylene) -ditetrazolium chloride (tetranitro-BT or TNBT) was synthesized. No extensive investigations or evaluations of the properties of this dye and its use in cytochemistry have been made until relatively recently in brief reports by Pearse and Hess (13), Rosa and Tsou (14), and Velardo and Rosa (22). The lastnamed workers indicated by illustrations the cytochemical use of T N B T in part of the female reproductive tract. Studies by Novikoff et al. (10) and others (5) have pointed out certain difficulties in using NBT for staining intracellular structures which are in 445 on Jne 6, 2017 D ow nladed fom Published March 1, 1963