Senescence-associated β-galactosidase histochemistry for the primate eye

PURPOSE. To develop a senescence-associated β-galactosidase histochemistry and bleaching protocol for the primate posterior pole. METHODS. Rhesus monkey eyes of different ages were enucleated after death, fixed in 4% paraformaldehyde for up to 16 hours, and cryoprotected using a graded sucrose infiltration technique. Ten-micrometer tissue sections were treated with β-galactosidase, pH 4 (lysosomal) or pH 6 (senescence- associated) activity, for various times. Bleaching of retinal pigment epithelial (RPE) cell and choroidal melanocyte pigment was performed after β-galactosidase histochemistry using 0.1% to 1% potassium permanganate incubation for 1 minute to 2 hours followed by 0.5% oxalic acid immersion. RESULTS. A 6-hour incubation with β-galactosidase, pH 4 or 6, demonstrated optimal staining of the RPE Uniform staining of the RPE for pH 4 β- galactosidase was seen in both young and old eyes. In contrast, senescence- associated β-galactosidase (pH 6) staining was seen in the RPE of 16- and 29-year-old, but not 1- and 2-year-old eyes. Senescence-associated β- galactosidase staining was evident in RPE cells adjacent to cuticular drusen. Optimal bleaching without loss of β-galactosidase staining was obtained using a 25-minute incubation with 0.05% permanganate. CONCLUSIONS. The senescence-associated β-galactosidase histochemistry assay, adapted for use in the primate posterior pole, showed staining of RPE cells in older eyes. Visualization of β-galactosidase activity in the RPE was enhanced by permanganate bleaching of melanin pigment. This technique could be valuable for identifying senescent RPE cells in human eyes.