Table 2:

Suggested Troubleshooting. Potential problems and solutions when using this method.

Problem	Solution
Sample comes out of agarose block during section	Work quickly with the molten agarose, blot the tissue gently but thoroughly, swirl the tissue in the molten agarose so that any residual PBS is mixed in with the agarose. Ensure there is enough agarose surrounding the tissue after trimming the block and that the block remains cold during sectioning.
Morphology artifacts	Use soft brushes, careful handling, slower vibratome blade advancing. Adjust fixation times. Adjust agarose % for embedding.
Thickness of section not accurate	We confirmed the vibratome section thickness is calibrated using Leica Vibrocheck. If the user does not have access to a vibratome calibrator, they can check the thickness by performing z-stacks on a confocal microscope and adjust the vibratome settings to compensate for differences in visualized thickness versus expected thickness. When using confocal imaging to assess section thickness, it is essential that the user imaging configuration has been validated with fluorescent bead standards
Low signal (broadly)	Use cold PFA and fix for no longer than 3–6 hours. Ensure Me0H is not warming up during post-fix. Keep isopropanol incubations at 50 seconds each, extending this step will reduce signal. Use fresh samples or store samples in accordance with the protocol notes to prevent signal decay.
Low signal (interior/middle of section)	Add 1% DMSO to staining buffer, increase rocker speed, perform Ab staining steps at room temp, or extend incubation time. Increase staining reagent concentration. Though not suitable for studies quantifying signal intensity, uneven staining across the thickness can be overcome using adaptive imaging where excitation laser power is adjusted throughout the acquisition to give uniform signal, or post processing algorithms, such as ImageJ/FIJI (Menu: Images→Adjust→Bleach Correction→Histogram Matching).
Distortions in z-dimension (broadly)	Adjust correction collar on objective lens or adjust coverslip thickness and confirm with fluorescent beads; or (not recommended for quantitative studies) correct z-step size post processing using calibration values determine by fluorescent microsphere standards.
Distortions in z-dimension (one-sided)	This is consistent with going over the working distance limits of the objective, where the objective begins pushing up against the coverslip and z-step size is no longer accurate. This can also potentially damage the objective. Use thinner coverslips (i.e. #0) or switch from a 60x oil objective to 40x oil for more working distance.
Sample cannot get in focus on high power objective	Use thin #0 coverslips. Ensure that the sample is flat and well-adhered on the coverslip throughout the clearing steps, taking care to transfer the samples gently from each solution. Gently handle the slides after sealing with nail polish. Though the sample is sealed between the coverslip and slide, if it gets detached from the coverslip this will result in less sample imageable within the working distance of the objective.
Computer crashes	Reduce bit-depth, the human eye cannot distinguish more than 250 shades of gray, thus even 8-bit images are suitable for most applications. Alternatively, sub-sample regions of interest. Commercial software, such as Nikon Elements or Bitplane lmaris, can handle large file sizes more efficiently.