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Finally, we design a new memory efficient solution for the specimen-level interior problem of cryoET, removing all associated artifacts.

The specimens for the OM and SEM observations were prepared using Nital (2 %) etching solution on the specimen's surface.

Fix tissues overnight at 4°C. 1. Prepare fresh 4% formaldehyde solution for tissue specimen dissection and fixation (refer to Table A1 for reagent and equipment details): a.

Prepare fresh 4% formaldehyde solution for tissue specimen dissection and fixation (refer to Table A1 for reagent and equipment details): a.

The specimens were placed into 10% formaldehyde solution for the latter histological examination.

A small amount of BCP solution was dropped on the specimen holder.

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After three washes with blocking solution for 5 min, specimens were incubated with the secondary antibody (Alexa Fluor 488 or 568, Invitrogen) for 2 h, at RT. FluoroJade C (Millipore, Darmstadt, Germany) was used to visualize all degenerating neurons.

These experimental data were used in combination with finite element solutions for all specimen geometries to determine a 'critical distance', a quantity or parameter determined from the stress distribution surrounding the notch in combination with fatigue limit stress data from unnotched specimens.

The operator loaded the two data sets for each solution of the same specimen into a workstation screen, scrolling the data sets in parallel with standard soft-tissue window settings (window width:700 HU; window center:140 HU).