A new video pachometer.

use in the posterior pole of the primate eye. The indigogenic method using X-Gal forms a granular precipitate that can be at least partially visualized in the presence of melanin pigment. Our procedure yields uniform staining of the RPE at pH 4.0, which •would be expected for lysosomal /3-galactosidase. It is of interest that the lysosomes of other cell types were much more weakly stained with this protocol. This finding undoubtedly reflects the very active lysosomal compartment of the RPE in comparison to other cell types in the retina/choroid. Staining at pH 4.0 for prolonged periods of time did reveal the presence of lysosomal /3-galactosidase in other cell types (data not shown). Staining for senescence-associated /3-galactosidase at pH 6 was uniformly negative in eyes from animals 1 or 2 years of age and yielded intermittent staining of the RPE monolayer in eyes from animals 16 and 29 years of age. It was especially interesting that RPE cells adjacent to cuticular drusen stained for senescence-associated /3-galactosidase. A rhesus macaque at 29 years is equivalent to an 85to 90-year-old human. 9 Quantitative data for the appearance of senescence-associated /3-galactosidase-positive cells as a function of chronological age or topographical distribution in the posterior pole were not provided in this brief study because of the very limited number of globes that were examined. Tissue fixation and embedment protocols proved to be a critical aspect of our method. We found, for example, that postmortem times to fixation in excess of 6 hours yielded RPE lysis and inconsistent staining results (data not shown). Our data suggest that both well-controlled fixation and cryoprotection improved the reliability of our results. These observations are consistent with comments in standard histochemistry texts on the sensitivity of lysosomal /3-galactosidase to aldehyde fixation. Fixation and tissue processing for the senescenceassociated /3-galactosidase activity at pH 6.0 may have identical or differing sensitivity to tissue-processing protocols. Currently it is not known how the pH 6.0 senescence-associated /3-galactosidase activity is related to the pH 4.0 lysosomal /3-galactosidase activity. These activities may be a function of identical or differing posttranslational modifications of the same gene product or may arise from different gene products. Our permanganate-bleaching protocol resulted in a method that might prove useful for applications other than our histochemical procedure. By extensively fixing sections after the enzyme histochemistry step, we were able to bleach a very substantial percentage of all pigment with the retention of both adequate morphology and chromogenic product. Because color-based alkaline phosphatase protocols for in situ hybridization and immunohistochemistr)' can use chromogens that are resistant to bleaching, our protocol may be directly applicable to these methods. This study has identified tissue processing as a very significant issue that must be carefully controlled in any planned studies of the human eye. It will be important next to apply these procedures to the human eye for an investigation of the presence of senescence-associated /3-galactosidase activity in the RPE.