For bisretinoid analysis carried out on adult zebrafish, three fish per strain were euthanized in tricaine (MS‐222) prior to eye dissection on ice under dim red light. Samples were snap frozen in liquid nitrogen and stored at −80°C. Animals had been dark adapted for 12 h prior to euthanasia. Bisretinoids were extracted from zebrafish eyes under red dim light. Briefly, six zebrafish eyes per each biological sample were washed with PBS and homogenized in 1 ml PBS. Chloroform/methanol (4 ml, 2:1, vol/vol) was added, and the samples were extracted with the addition of 4 ml of chloroform and 3 mL of dH2O, followed by centrifugation at 1000 g for 10 min. Extraction was repeated with the addition of 4 ml of chloroform. Organic phases were pooled, filtered, dried under a stream of argon, and redissolved in 100 μl of 2‐propanol. Bisretinoid extracts were analyzed by normal‐phase HPLC with a silica column (Zorbax‐Sil 5 μm, 250 × 4.6 mm; Agilent Technologies) as previously described in Radu et al (IOVS 2008).34 The identity of each bisretinoid peak was confirmed by online spectral analysis. A2PE data were presented as means with standard deviation of four biological samples for each genotype.
Zorbax sil 5 μm
Manufactured by Agilent Technologies
Zorbax‐Sil 5 μm is a silica‐based packing material used in high‐performance liquid chromatography (HPLC) applications. It has a particle size of 5 micrometers and is designed for analytical separations.
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3 protocols using zorbax sil 5 μm
1
Bisretinoid Analysis in Zebrafish Eyes
2
Retinoid Analysis in AdipoR1 Knockout Mice
3
Retinoid Analysis in Mice
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Overnight dark-adapted wild-type (n=4) and AdipoR1−/− (n=4) mice (3–4 months of age) were anaesthetized with an intraperitoneal injection of ketamine and xylazine. Pupils were dilated with 1% (w/v) atropine sulfate in saline solution. Two mice of each genotype were exposed to light at 1,000 lux for 10 min; the remaining mice were maintained in darkness. Mice were killed by cervical dislocation under anaesthesia and eyes enucleated. After removing the anterior segment, eyecups were homogenized in 20 mM HEPES buffer containing 0.1% SDS and hydroxylamine, and retinoids extracted with hexane under dim red light (Kodak Wratten 1A). Total retinoids, all-trans-retinyl esters and 11-cis retinaldehyde from the dark-adapted and light-exposed mice were measured by normal-phase HPLC (Agilent 1100 liquid chromatograph) equipped with a ultraviolet photodiode-array detector. Retinoids in the samples were separated by gradient elution of the mobile phase (0.2–10% dioxane in hexane, 2 ml min−1 flow rate) on a silica column (Zorbax-Sil 5 μm, 250 × 4.6 mm, Agilent Technologies). Identified peaks were confirmed by spectral analysis and co-elution with authentic retinoid standards.
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