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The membrane was blocked with 5% milk in TBST (20 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.05% Tween-20), then incubated with the anti-GluK2 monoclonal antibody (2000-fold dilution, 04-921, Sigma-Aldrich), followed by horseradis peroxidase (HRP)-conjugated anti-rabbit antibody (10,000-fold dilution, ab97051, abcam)

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Melatonin alone had no effect, and there was little interaction between IF and melatonin except for their effects on adipocyte area and number, the abundance of Bacteroidetes and Akkermansia , and the intestinal metabolites alanine, valine and isoleucine

Carefully remove any air bubbles from the syringe by flicking it gently and pushing the plunger until a tiny drop of solvent appears at the needle tip