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In a retrospective study, it was observed that subjects receiving doses of metformin higher than 2000 mg/day or for more than 4 years had low levels of vitamin B 12 [18]

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Cells were extensively washed with PBS, fixed in 4% PFA for 15 min, permeabilized and blocked with 0.01% saponin/10% normal goat/PBS serum and stained with the primary antibodies in the same blocking solution in a humid chamber

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