Table 1.

Troubleshooting Table

Step	Problem	Possible reason	Solutions
Procedure 1 step 4.	Poor or uneven expansion, sample tearing	Inadequate denaturation or digestion of the sample may cause sample tearing or under-expanded regions in the nuclei distribution (see Fig. 10).	Extend the denaturation period and/or repeat the denaturation step at a higher temperature. In the case of enzymatic digestion, an addition of another category of enzyme (such as elastase) may help with digestion49,50 (see Fig. 10).
Procedure 1 steps 3, 25, 26 OR Procedure 2 step 16	No signal	Lack of signal can arise due to several reasons:
		Inappropriate microscope settings	Check that the microscope settings are appropriate to the fluorophores with a suitable positive control.
		Issues with sample gelation	Make sure that the hydrogel polymerization is done on the coverslip side coated with cells as it can be easy to lose track of which side of a coverslip the cells are adhered to (Fig. 11b). Make sure that the sample is positioned as close to the surface of the gel as possible in order to be within the working distance of the objective lens.
		Error with sample mounting	It is possible that the gel is upside down, and the surface of the hydrogel containing the sample is outside the working distance of the objective lens (Fig. 11c). Test this by imaging the other surface of the gel.
		Fluorophore bleaching	The NaIO4 oxidation step will chemically bleach many fluorophores, and adequate removal of NaIO4 is required prior to the hydrazide dye incubation. Repeat the procedure with more thorough washing. Importantly, the hydrazide labeling step should be performed before the NHS-dye coupling reaction, or any other fluorescent labeling reactions.
Procedure 1 steps 10, 12, 17, 25, 26 OR Procedure 2 steps 2, 3, 4, 16	Dim Signal	Dim signals can arise for several reasons:
		Reagent degradation can result in an inefficient reaction.	If the carbohydrate stain is dim, it could indicate that NaCNBH3, which can become deactivated with prolonged exposure to moist air, has been hydrolyzed, leaving the hydrazide-dye conjugated to the specimen by a relatively labile hydrazone bond. If the amine channel is dim, it could indicate that the NHS groups on the NHS-dye have been hydrolyzed and thus the dyes were not efficiently coupled to the sample. In either of these cases, researchers can repeat the procedure using fresh aliquots or newly purchased reagents. In addition, proper storage can help slow down the degradation of these reagents (refer to the Reagents set-up section).
		Insufficient dye concentration	The sample staining intensity can be tuned to produce brighter signals by increasing the concentration of the reactive fluorophores used in the labeling reactions.
		Lack of sample exposure to chemicals	With respect to the hydrogel-embedded samples, make sure that the hydrogel surface containing the sample is placed face-up in a container during the labeling reaction, or the dyes may have a harder time accessing the sample resulting in dim signals (Fig. 11a).
		Incorrect objective lens	It is essential to carefully choose the correct objective lens for a given type of sample: water-immersion objective lenses are well-suited to imaging specimens in hydrogels, whereas oil-immersion lenses are well-suited to imaging organic cleared oil refractive index matched EC specimens. The use of a mismatched immersion lens may lead to optical aberrations and loss of signal at substantial depths.
		Inner filter effect	See advice given for an uneven Signal.
Procedure 1 step 26 OR Procedure 2 step 16	Uneven Signal	Uneven signals mostly occur following labeling of thick samples and could be a result of excess dye during a labeling reaction. A thick sample can be labeled so strongly at the surface that the excitation light in the microscope is substantially attenuated within the interior of the sample (“inner filter effect”), producing a very bright surface but an apparent dimmer interior, even if the sample itself is in fact labeled uniformly.	Section the hydrogel or the thick sample and image the cross-section to assess the sample uniformity without this complication (see Fig. 12). If the stain is indeed uniform, stain a new sample with a more dilute dye to produce a dimmer stain that avoids this inner filter effect (Fig. 12). If the stain is in fact enriched at the surface, consider preparing the NHS-dye stock in ~100 mM MES buffer at pH 6.0 to slow down the hydrolysis and coupling reactions while also providing more time for the labeling reaction and dye penetration. In addition, to aid in the penetration of the dye through the expanded samples which are embedded in negatively charged hydrogel, consider using NaOAc/NaCl buffer solution to help allow charged reactants to enter the negatively charged hydrogel gel. Also, make sure that the bottom surface of the tissue sample is not attached to the surface of the container during incubation (Fig. 11a) and to ensure continuous sample motion during gentle agitation. Using a lower concentration of dye may also help improve the uniformity of labeling, but possibly at the cost of decreasing the brightness of the stain.
Procedure 1 steps 15, 17 OR Procedure 2 Steps 2, 4	Overly Bright Signals	Crosstalk from an overly bright signal in a channel could swamp a weaker signal from a different spectral channel. In addition, an overly bright signal can produce inner filter effect. (see Uneven Signal) Overly bright signals can arise due to several reasons:
		Insufficient washing could prevent unconjugated dyes from being removed from the sample resulting in very bright signals.	Washing the sample extensively can fix this problem and improve contrast.
		If the dye concentration is too high, an overly bright sample may be produced.	Lowering the concentration of dye(s) in the coupling reaction(s) can help tune the signal.
Procedure 2 step 7–11	Poor Transparency in Refractive Index Matched Samples	Improper or insufficient tissue clearing can arise for several reasons:
		Inadequate dehydration prior to incubation with ethyl cinnamate (EC) or other common refractive index oil.	Thorough dehydration is crucial to achieve a uniform refractive index match throughout the entire specimen thickness and extending the dehydration periods at each stage may help improve the clarity.
		Insufficient incubation with EC	Refresh the EC a couple of times, followed by waiting ~30–60 min or more (depending on the size of the tissue) for the tissue to become fully cleared and index-matched.