Effect of Buffer Storage on Fine Structure and Catalase Cytochemistry of Peroxisomes

Peroxisomes are morphologically characterized by a single limiting membrane and a finely granular matrix, and some of them contain crystalline nucleoids (1, 2). Application of the alkaline 3,3'-diaminobenzidine (DAB) method, which visualizes the peroxidatic activity of catalase (3, 4) in normal liver, has revealed electron-dense reaction product in the matrix of peroxisomes, but none in the cytoplasm (5, 6). Legg and Wood and associates, on the other hand, have described evidence of staining of ribosomes and the endoplasmic reticu-lum membranes adjacent to peroxisomes in liver of normal and treated rats in conditions associated with increased de novo synthesis of catalase (7-10). Although these authors raised the possibility that diffusion artifacts might cause the ribosomal staining , they considered it unlikely since "under all conditions used, microbodies with sharp localiza-tion of reaction product were present in the same sections that showed microbodies with adjacent ribosomal staining" (7). Some of these observations were also corroborated in preliminary studies , from ours as well as various other laboratories (11-15). Nevertheless, Novikoff et al., in discussing the various causes of diffusion artifacts in DAB cytochemistry (16), concluded that the ribosomal staining adjacent to peroxisomes is due to diffusion of oxidized DAB, which is generated within the peroxisomes and subsequently diffuses out and is adsorbed on ribosomes (16, 17). Recently, we studied the various causes of diffusion artifacts in the cytochemistry of catalase and demonstrated that indeed the ribosomal staining is due to diffusion of catalase rather than oxidized DAB (18). Such diffusion occurs in the course of rinsing or storage of tissue sections in buffer after aldehyde fixation and before incubation in DAB medium (18). The present communication deals with two questions: (a) what is the effect of buffer storage upon the fine structure of peroxisomes; and (b) does exposure to buffer affect all peroxisomes uniformly, or is there a heterogeneous response, with some peroxisomes exhibiting diffusion and other adjacent particles within the same cell exhibiting none? MATERIALS AND METHODS Animals Male adult albino rats of the Charles River strain (CDR) weighing 250-350 g and fed a normal diet and water ad libitum were used. The animals were fasted for 16 h before sacrifice in order to decrease the content of hepatic glycogen. Fixation All livers were fixed by perfusion through the portal vein as described previously (5). The fixative contained 2.5% distilled glutaraldehyde (Ladd Industries, Burling-ton, Vt.) in 0.1 M cacodylate buffer pH …