Table 5 |.

Troubleshooting Table

Step	Problem	Possible reason	Solution
1	Coverslip can’t be removed from slide (for mounted tissue slides)	Insufficient xylene incubation time when removing the mounting medium	Longer incubation time with xylene
2	Tissue detaches from the slide	(1) The tissue slide is not baked (2) Super-frost glass slides were not used (3) Poor quality of specimen	Use super-frost glass slides for holding tissue sections. Bake the tissue slide at 60 °C for 10 min to enhance adhesion
3–4	Dim, uneven or absent fluorescence signal (pre-expanded specimens)	Sub-optimal immunostaining conditions	Optimize immunostaining parameters: temperature, buffer composition and duration for antigen retrieval, the concentration of antibodies, incubation times for staining, the antibodies used.
7	No gel is formed	(1) Low concentration of APS and/or TEMED (2) The gelling solution was not replaced with a freshly prepared one, at the indicated time. (3) The crosslinker was not added or its concentration is too low (i.e., much less than 0.1%)	(1) Add the correct amount of APS and TEMED. (2) Ensure the gelling solution is replaced with fresh solution after the initial incubation in the gelling solution at 4 °C. We observed that the gelling reaction becomes inconsistent if the incubation at 4 °C lasts longer than 2 hours without replacement of the gelling solution. (3) Make sure the final concentration of crosslinker is more than 0.1%
7	The gel appears to be highly fragile and viscous.	(1) Insufficient time for polymerization. (2) The 4-HT concentration is too high. (3) The quality of sodium acrylate is low.	(1) Ensure the incubation time is the correct one. If necessary, try a longer incubation time (e.g. 3 hours) to complete the gelling reaction. (2) Add the correct amount of 4-HT. If the issue persists, try to reduce the amount of 4-HT by half and perform the gelling process. Note that reducing 4-HT concentration might result in premature gelling during the pre-gelation incubation in the fridge. (3) Get a new batch of sodium acrylate Optionally, the mechanical strength of gel can be improved by increasing the final concentration of sodium acrylate to 12 g/100 ml and the final concentration of acrylamide to 3.5 g/100 ml while maintaining the rest of the ingredients at the same concentrations indicated in Table 1.
10	The gel is still adhered to the slide	Insufficient digestion	Incubate longer with proteinase K solution OR increase the proteinase K concentration; try manually removing the gel from the slide (Step 10B).
10	The gel appears to be warped or curved in solution after digestion	This is caused by insufficient digestion of the gel due to: (1) The concentration of AcX is too high (2) Sub-optimal digestion conditions	(1) Lower the concentration of AcX by half. (2) First, make sure the incubation temperature is 60 °C; second, try doubling the proteinase K concentration to see if the result is improved; finally, try to increase the incubation time: we recommend examination of the physical appearance of the gel every 30min during digestion; as a rule of thumb, if the gel appears to be flat and transparent, that suggests a successful digestion and further incubation is not necessary.
11	The gel gets sucked into the pipette tip and damaged.	(3) The specimen is transparent and hard to track in the aqueous solutions.	(3) Gently shake the specimen chamber and look for the water-gel boundary. Alternatively, illuminate the chamber with a white light LED from various angles to help locate the specimen.
12	The expansion factor is less than 4 fold	(1) Insufficient dialysis with pure water Using salt-containing buffer instead of water for dialysis	Make sure sufficient buffer exchange with pure water has occurred; make sure fresh gelling solution was used in the gelation step
12	The gel is bigger than the expansion chamber	(2) Gel piece is too big to fit in the chamber	Trim the gel and carefully move a part of it into another expansion chamber
14	Dim or absent fluorescence signal after expansion	(1) Less suitable fluorophores were used, such as cyanine dyes Cy5, Cy3, and Alexa Fluor 647 (2) The gel may be in the wrong orientation (1) Overdigestion by proteinase K	(1) Use the recommended fluorophores (Alexa 488, Alexa 546 and Atto 647N or CF633) (2) Most high-magnification objectives do not have long enough working distance to image through an entire gel. Use paint brush to flip the gel so that the specimen faces the objective. (3) Shorten the digestion time by half or reduce the proteinase K concentration by half and try again until the optimal digestion condition is found.
14	Focus is drifting	(1) The specimen is not mounted properly (2) Temperature of specimens and optical components of the microscopes are different	(1) Mount the specimen with agarose (2) Wait until the temperature of the whole system is the same (3) Treat the glass surface with poly-L-lysine solution before placing the specimen.
14	Unable to locate the same field of view taken before expansion.	(1) Expansion brings in more detailed tissue images but sometime this additional information can be confusing. In addition, the expanded specimen may be in a different orientation than the one of the pre-expansion image.	First, use low magnification objectives to image the whole tissue section or at least a large portion of the entire specimen. Then, locate the matched field of view of the expanded specimen by comparing the two low-magnification tissue images taken before and after expansion. Finally, center the stage to the matched field of view and acquire images with high-magnification objectives.
14	Tissue slice is not completely flat in the gel; even minute waves in the tissue slice are amplified with expansion and can become problematic by reducing imaging quality and throughput	Tissue slice partially detached during the process. Tissue slice is not uniformly adhered to the slide.	(1) Use super-frost glass slide to deposit tissue slices. (2) Bake the tissue slide in 60C for 10 minutes before the specimen formatting process. (3) Gentle handling to minimize disturbance to the tissue section