Zebrafish were euthanized by the rapid cooling method [10 (link)] (immersion in ice-cold water at 2–4 °C for 10 min) after behavioral testing. The brains were precisely dissected for biochemical analyses. For this, the brains were homogenized in ice 0.1 M potassium phosphate buffer (pH 7.4), 1.15% KCl with Mikro-Dismembrator U mill (Sartorius, New York, NY, USA) equipped with 3 mm diameter magnetic balls (Sartorius Stedim Biotech GmbH, Goettingen, Germany). Samples were centrifuged at 960× g for 15 min and the supernatant was used for the estimation of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) specific activities, reduced glutathione (GSH), protein carbonyl and malondialdehyde (MDA) levels, following the methods described in detail by Boiangiu et al. [45 (link)].
Mikro dismembrator u mill
Manufactured by Sartorius
Sourced in United States
The Mikro-Dismembrator U mill is a high-performance laboratory mill designed for the efficient grinding and homogenization of small sample quantities. It utilizes a tumbling motion to disintegrate samples into fine powders, ensuring thorough and reproducible sample preparation.
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4 protocols using mikro dismembrator u mill
1
Zebrafish Brain Biochemical Analysis
2
Brain Tissue Homogenization and Biochemical Analysis
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The collected brain tissues were smoothly homogenized (1:10 ratio, w/v) in cold 0.1 M potassium phosphate buffer (pH 7.4) containing 1.15% KCl. The homogenization was carried out for 1 min at 1000 rpm using a Mikro-Dismembrator U mill (Sartorius, New York, NY, USA) equipped with 3 mm diameter magnetic balls (Sartorius Stedim Biotech GmbH, Goettingen, Germany). The resulting homogenate was then subjected to centrifugation for 15 min at 14,000 rpm. The supernatant was collected for further analysis. This included the measurement of the total soluble protein content and the evaluation of the activities of specific enzymes such as acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX); glutathione (GSH); malondialdehyde (MDA) and carbonylated proteins. All procedures were carried out in conformity with the applicable rules and regulations as mentioned in previous studies [60 (link)].
3
Brain Tissue Analysis Protocol
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Shortly after performing the in vivo tasks, the fish were euthanized by rapid cooling, as this method does not cause biochemical or physiological changes that could prevent post-mortem analysis [51 (link)]. Subsequently, the fish were dissected using the procedure described by Gupta and Mullins [52 (link)], and whole brains were collected for biochemical analysis. A pool of two brains was considered one independent sample. The brain tissues were gently homogenized (1:10 ratio, w/v) for 1 min at 1000 rpm in cold 0.1 M potassium phosphate buffer (pH 7.4), 1.15% KCl with the Mikro-Dismembrator U mill (Sartorius, New York, NY, USA) equipped with 3 mm diameter magnetic balls (Sartorius Stedim Biotech GmbH, Goettingen, Germany). The homogenate was centrifuged for 15 min at 14,000 rpm and the supernatant was used to determine the total content of soluble protein, to assess the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and acetylcholinesterase- (AChE)-specific activities and to estimate the level of reduced glutathione (GSH), malondialdehyde (MDA) and carbonylated proteins.
4
Zebrafish Brain Biochemical Analysis
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Once the behavioral tasks were finished, the zebrafish were euthanized by rapid cooling. It is known that this method avoids the biochemical and physiological alterations that could prevent post-mortem analysis [49 (link)]. The animals were immersed in ice-cold water (2–4 °C) until no opercular or tail movements were observed. Subsequently, the whole brain was carefully dissected according to the procedure described by Gupta and Mullins [50 (link)] and collected for biochemical analysis. Two brains were pooled and considered as an independent sample. The brains were homogenized (1:10 ratio, w/v) in ice-cold 0.1 M potassium phosphate buffer with 1.15% KCl (pH 7.4), for 1 min at 1000 rpm using the Mikro-Dismembrator U mill (Sartorius, New York, NY, USA) equipped with 3 mm diameter magnetic balls (Sartorius Stedim Biotech GmbH, Goettingen, Germany). Then, the crude homogenates were centrifuged for 15 min at 14,000 rpm and 4 °C, and the supernatants were further used to measure the total content of soluble proteins (bicinchoninic acid assay) [51 (link)], the activities of SOD (EC 1.15.1.1) [52 (link)], CAT (EC 1.11.1.6) [53 (link)], GPX (EC 1.11.1.9) [54 (link)], AChE (E.C. 3.1.1.7) [55 (link)] and the levels of MDA [56 (link)] and carbonylated proteins [57 (link)].
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