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Under HG conditions, the mitochondrial respiratory chain releases excess ROS to the cytoplasm to signal hypoxia, triggering mitochondrial depolarization damage

To prevent this, use the smallest gauge needle practical for drawing doses (29-31 gauge insulin needles), always insert the needle straight (not at an angle), and try to puncture the same spot on the stopper each time rather than creating new holes across the surface

After washing with 50% EtOH, the sections were coverslipped

If tolerated, the documented approach is to stay at the current step for the full interval before the next scheduled escalation not to leapfrog steps

[4] Minich, D