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 language | English (US) |
|---|---|
| Pages (from-to) | 1590-1593 |
| Number of pages | 4 |
| Journal | Investigative Ophthalmology and Visual Science |
| Volume | 40 |
| Issue number | 7 |
| State | Published - 1999 |
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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 journal › Article
}
TY - JOUR
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
Y1 - 1999
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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M3 - Article
C2 - 10359342
AN - SCOPUS:0032978077
VL - 40
SP - 1590
EP - 1593
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
SN - 0146-0404
IS - 7
ER -