June 1999
Volume 40, Issue 7
Free
Articles  |   June 1999
Senescence-associated beta-galactosidase histochemistry for the primate eye.
Author Affiliations
  • K Mishima
    Department of Ophthalmology, University of California, Davis 95616-8794, USA.
  • J T Handa
    Department of Ophthalmology, University of California, Davis 95616-8794, USA.
  • A Aotaki-Keen
    Department of Ophthalmology, University of California, Davis 95616-8794, USA.
  • G A Lutty
    Department of Ophthalmology, University of California, Davis 95616-8794, USA.
  • L S Morse
    Department of Ophthalmology, University of California, Davis 95616-8794, USA.
  • L M Hjelmeland
    Department of Ophthalmology, University of California, Davis 95616-8794, USA.
Investigative Ophthalmology & Visual Science June 1999, Vol.40, 1590-1593. doi:
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      K Mishima, J T Handa, A Aotaki-Keen, G A Lutty, L S Morse, L M Hjelmeland; Senescence-associated beta-galactosidase histochemistry for the primate eye.. Invest. Ophthalmol. Vis. Sci. 1999;40(7):1590-1593.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

PURPOSE: To develop a senescence-associated beta-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 beta-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 beta-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 beta-galactosidase, pH 4 or 6, demonstrated optimal staining of the RPE. Uniform staining of the RPE for pH 4 beta-galactosidase was seen in both young and old eyes. In contrast, senescence-associated beta-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 beta-galactosidase staining was evident in RPE cells adjacent to cuticular drusen. Optimal bleaching without loss of beta-galactosidase staining was obtained using a 25-minute incubation with 0.05% permanganate. CONCLUSIONS: The senescence-associated beta-galactosidase histochemistry assay, adapted for use in the primate posterior pole, showed staining of RPE cells in older eyes. Visualization of beta-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.

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