Immunofluorescence was performed for both whole mounts and transverse sections of the retina.45 (link) Briefly, retinal cryosections were blocked in 0.5% Triton X-100 and 5% bovine serum albumin in PBS for 1 h at room temperature. Afterward, sections were incubated overnight at 4°C with primary antibodies for anti-IBA1+ (019–19741, 1:1000, Wako, Tokyo, Japan), anti-Rhodopsin (MAB5356, 1:500, Merck, Darmstadt, German), anti-Cone arrestin (AB15282, 1:1000, Merck, Darmstadt, German), and anti-MFRP (AF3445, 1:40, R&D, Minnesota, USA). After washing three times with PBST (1% Tween 20 in PBS), samples were incubated with Alexa 594 (ab150080/ab150116, 1:1000, Abcam, Cambridge, MA, USA) or FITC (PA1-28734, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA) secondary antibodies for 1 h. Subsequently, sections were washed three times with PBS and stained with DAPI. Images were captured using an Olympus FV3000 confocal microscope. The number of infiltrated microglia and GFP density in the RPE layer were quantified by IMARIS software.
Af3445
Manufactured by R&D Systems
Sourced in United States
AF3445 is a laboratory instrument designed for the analysis and separation of biological samples. It utilizes advanced technology to facilitate precise and efficient sample processing. The core function of this product is to enable researchers to conduct critical analyses and experiments in a reliable and controlled environment.
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Lab products found in correlation
2 protocols using af3445
1
Retinal Immunofluorescence Staining Protocol
2
Immunohistochemistry of Murine RPE
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Eyes were enucleated from P7 and P22 mice, and cornea, lens, optic nerve, and retina tissues were removed. At least 3 animals/eyes per genotype per time point were used. P7 eyes were treated for 5 minutes with PBTx to aid in the separation of the retina from the RPE. RPE was fixed for 45 minutes in phosphate buffered 4% paraformaldehyde. After fixation, RPE samples were rinsed in PBS and incubated in phosphate buffered 3% H2O2 for 48 hours at 4°C to quench the RPE autofluorescence. RPE samples were washed in PBS and blocked in 10% Normal Goat Serum (NGS), 1% BSA in PBTx for 2 hours. Primary antibody incubation occurred overnight at 4°C with the antibodies and dilutions to the following antigens: TMEM98 (1:500, 14731-1-AP, Proteintech), MFRP (1:500, af3445, R&D Biosystems, Minneapolis, MN, USA). RPE samples were washed in PBS and incubated with Alexa-conjugated fluorescent secondary antibodies for 2 hours at 22°C. RPE samples were rinsed in PBS and the nuclei were stained with DAPI. A series of 3–4 radial cuts were made to flatten the RPE and underlying scleral and samples were mounted with Prolong Gold Antifade. Images were taken on a Leica SP5 confocal microscope or Olympus BX-51 epifluorescence microscope.
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