Senescence-associated β-galactosidase histochemistry for the primate eye

Kazuaki Mishima, James T. Handa, Amy Aotaki-Keen, Gerard A. Lutty, Lawrence S Morse, Leonard M Hjelmeland

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)1590-1593
Number of pages4
JournalInvestigative Ophthalmology and Visual Science
Volume40
Issue number7
StatePublished - 1999

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Galactosidases
Retinal Pigments
Primates
Staining and Labeling
Epithelial Cells
Potassium Permanganate
Oxalic Acid
Melanocytes
Melanins
Immersion
Macaca mulatta
Sucrose

ASJC Scopus subject areas

  • Ophthalmology

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Senescence-associated β-galactosidase histochemistry for the primate eye. / Mishima, Kazuaki; Handa, James T.; Aotaki-Keen, Amy; Lutty, Gerard A.; Morse, Lawrence S; Hjelmeland, Leonard M.

In: Investigative Ophthalmology and Visual Science, Vol. 40, No. 7, 1999, p. 1590-1593.

Research output: Contribution to journalArticle

Mishima, Kazuaki ; Handa, James T. ; Aotaki-Keen, Amy ; Lutty, Gerard A. ; Morse, Lawrence S ; Hjelmeland, Leonard M. / Senescence-associated β-galactosidase histochemistry for the primate eye. In: Investigative Ophthalmology and Visual Science. 1999 ; Vol. 40, No. 7. pp. 1590-1593.
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title = "Senescence-associated β-galactosidase histochemistry for the primate eye",
abstract = "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.",
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T1 - Senescence-associated β-galactosidase histochemistry for the primate eye

AU - Mishima, Kazuaki

AU - Handa, James T.

AU - Aotaki-Keen, Amy

AU - Lutty, Gerard A.

AU - Morse, Lawrence S

AU - Hjelmeland, Leonard M

PY - 1999

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N2 - 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.

AB - 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.

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