Whole eyes without cornea and lens, dissected neural retina, or dissected posterior eyecups enriched in RPE and choroid were lysed in ice-cold HNTG lysis buffer (50 mM HEPES, pH 7.4, NaCl 150 mM, 10% glycerol, and 1% Triton X-100) supplemented with 1 mM phenylmethylsulfonyl fluoride and 1% protease inhibitor cocktail. Cleared lysates were subjected to standard SDS-PAGE, immunoblotting, and signal detection on autoradiography film using enhanced chemiluminescence (Lightning Plus; Perkin Elmer, Shelton, CT, USA). Primary antibodies were to: rhodopsin B630, blue opsin (Millipore-Sigma; #5407), red/green opsin (Millipore-Sigma; #5405), GAPDH (Genetex, Irvine, CA, USA; #GTX100118), gal-3, MerTK (R&D Systems; #AF591), RPE65 (Genetex; #GTX103472), and anti-PSD95 (Cell Signaling, Danvers, MA, USA; #S4507). Image-J software was used to quantify relative densities of bands.
Rpe65
Manufactured by GeneTex
Sourced in United States
RPE65 is a gene that provides instructions for making a protein called retinoid isomerohydrolase, which is essential for the visual cycle. This protein plays a critical role in the conversion of all-trans-retinol to 11-cis-retinol, a key step in the regeneration of the light-sensitive pigment in the retina.
Automatically generated - may contain errors
Lab products found in correlation
Mertk, by R&D Systems (1 mentions)
Anti psd95, by Cell Signaling Technology (1 mentions)
Gal 3, by R&D Systems (1 mentions)
Gapdh, by GeneTex (1 mentions)
Porin, by Cell Signaling Technology (1 mentions)
Psd 95, by Merck Group (1 mentions)
Akt and phospho akt ser473, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
Imagequant tl 7, by GE Healthcare (1 mentions)
Ab195352, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
F4 80, by Bio-Rad (1 mentions)
Hmga2, by Cell Signaling Technology (1 mentions)
Tcs sp8, by Leica (1 mentions)
Mca497, by Bio-Rad (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab2033, by Merck Group (1 mentions)
4 protocols using rpe65
1
Retinal Protein Extraction and Analysis
2
Immunoblotting Analysis of Retinal Proteins
3
Retinal Protein Expression Analysis
4
Quantifying Subretinal Fibrosis Volume
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Immunofluorescence assays were performed with retinal sections, RPE–choroid flatmounts, or primary RPE cells in 24-well slide chambers. Primary antibodies were used for staining against E-cadherin (3195, Cell Signaling Technology), N-cadherin (13116, Cell Signaling Technology), α-SMA (ab5694, Abcam), RPE65 (GTX13826, GeneTex), METTL3 (ab195352, Abcam), IB4 (DL-1207, Vector Laboratories), F4/80 (MCA497, Abd Serotec), Alexa FluorTM 488 Phalloidin (A12379, Thermo Fisher Scientific), Ki67 (ab15580, Abcam), HMGA2 (8179, Cell Signaling Technology), and GFP (ab290, Abcam).
To evaluate the volume of subretinal fibrosis, RPE–choroid flatmounts were stained with the antibody against Fibronectin (AB2033, Merck Millipore) on Day 28 after laser injury. The volume analysis was performed as previously described (Ye et al., 2015 (link)). Fibronectin was visualized using a confocal laser scanning microscope (TCS SP8; Leica Biosystems). Horizontal optical sections were taken at 1 µm intervals from the top to the bottom of the fibrosis lesion. The area of Fibronectin-positive lesion on each layer was measured using ImageJ software. The total area of each horizontal section was used as an index of Fibronectin volume. All laser spots in each eye were measured.
To evaluate the volume of subretinal fibrosis, RPE–choroid flatmounts were stained with the antibody against Fibronectin (AB2033, Merck Millipore) on Day 28 after laser injury. The volume analysis was performed as previously described (Ye et al., 2015 (link)). Fibronectin was visualized using a confocal laser scanning microscope (TCS SP8; Leica Biosystems). Horizontal optical sections were taken at 1 µm intervals from the top to the bottom of the fibrosis lesion. The area of Fibronectin-positive lesion on each layer was measured using ImageJ software. The total area of each horizontal section was used as an index of Fibronectin volume. All laser spots in each eye were measured.
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