Table 1.

Troubleshooting the Protocol

Step	Potential problem	Solution
Steps 1‐10	Cells are dead (e.g., appearing as round, non‐adherent cells) or contaminated, or other problems associated with cell culture occur	Learn and practice aseptic techniques. Please refer to Phelan, (2007) for detailed instructions on aseptic techniques. We also found video resources, such as Cell Culture Basics (https://www.thermofisher.com/us/en/home/references/gibco‐cell‐culture‐basics.html), to be educational and useful.
Steps 11‐14	Immunostained microtubules, before expansion, show discontinuous and/or distorted morphologies in step 24	Distorted or discontinuous morphologies of microtubules suggest failed fixation steps. We suggest that the users use the fixative vendors recommended in this protocol. Always use fresh fixatives from the ampules provided by the manufacturer; these ampules are intended for one‐time use after opening. The microtubule fixation solution (see recipe) must be used freshly and cannot be stored. Measure the pH of the PBS solution to confirm that the value is ∼pH 7.4. If the above troubleshooting attempts fail, discard all solutions and purchase new ones from the recommended vendors.
Steps 11‐28	Liquid aspiration from a 24‐well plate damages cells on the coverslip	When aspirating liquid from a well, use one hand to hold the 24‐well plate and the other to hold the pipet. First push and hold the pipet plunger to dispense air out of the pipet tip so that the pipet is ready for drawing up liquid. Then, carefully move the pipet tip to the bottom edge of the well, and carefully tilt the plate ∼15° toward the pipet tip. Finally, slowly release the plunger to draw the liquid into the pipet tip. Do not aspirate the liquid directly from the cell layer, as that might damage the cells.
	Cells dry out between steps	To prevent drying of cells, we recommend that beginners simultaneously handle no more than two wells at a time, to minimize the time between liquid aspiration and addition steps.
Step 13	Immunostained microtubules, before expansion, show significant background fluorescence signals in step 24	The sodium borohydride reduction solution (see recipe), used to quench the fixatives and reduce background fluorescence, must be made fresh before use. Sodium borohydride, when fresh, should generate hydrogen gas bubbles when dissolved in PBS. If no hydrogen gas bubbles observed, use a new bottle of sodium borohydride.
Steps 15‐24	Immunostained microtubules, before expansion, show very weak immunostaining signals in step 24	Antibodies must be stored per manufacturer's instructions to ensure good quality. Secondary antibodies must be stored in the dark to prevent dye photobleaching. Use brightfield imaging to inspect the morphology of the cells to confirm successful fixation. If fixation is successful, the likely cause of weak immunostaining signals is a bad lot of antibodies. Replace both primary and secondary antibodies with newly purchased ones using the catalog numbers and vendors provided in this protocol.
Step 26	Immunostained microtubules show good imaging outcomes before expansion in step 24, but very weak or even no fluorescence signals after expansion in step 54	AcX, which crosslinks proteins to the hydrogel network, is an NHS ester and is prone to degradation by hydration. Therefore, AcX powder must be stored in a container with drying reagents (e.g., Drierite) at −20°C. AcX must be dissolved in anhydrous DMSO rather than regular DMSO, and the aliquoted AcX solution must be stored in a desiccating container at −20°C. Also, measure the pH of the PBS solution to confirm that the value is around pH 7.4, as high pH (> 9) will quickly hydrolyze AcX before it reacts with proteins. If the above troubleshooting attempts fail, discard all AcX solutions and purchase new materials from the recommended vendors.
Steps 29‐30	Gelation solution gels prematurely before step 37	The speed of polymerization significantly increases at higher temperature (e.g., at human body temperature of ∼37°C) or with high concentrations of APS and TEMED. To prevent premature gelation, first, all solutions (Stock X, APS, and TEMED) must be chilled on ice before mixing. APS, the radical polymerization initiator, must be added last. The gelation solution must be immediately placed back on ice after adding APS and vortexing. Keep the gelation solution on ice when transferring the solution to the 24‐well plate in step 30. The 24‐well plate must be kept on ice during the 5‐min incubation period in step 30. If the above instructions were strictly followed, but the gelation solution still prematurely gelled, it is possible that the concentration of the APS stock solution was mistakenly high. Make a fresh APS stock solution and redo the experiment.
Step 31	Coverslips or glass slides are broken during chamber construction	Use forceps to carefully pick up and discard broken coverslips or glass slides into a sharp waste container. Be very careful and avoid getting wounded by the broken glass. Then, redo step 31 with new glass slides and coverslips.
Steps 32‐34	Cell culture coverslip dries before the chamber is filled with gelation solution in step 35	Steps 33 and 34 must be finished within 2 min so that the cells will not dry out. If the user finds these steps to be demanding, we suggest practicing them with clean cell culture coverslips placed in a clean 24‐well plate before performing the actual protocol.
	Cell culture coverslip is broken	Use forceps to carefully pick up and discard broken coverslips into a biological sharp waste container. Be very careful and avoid getting wounded by the broken glass. Then, redo the experiments.
Step 35	Air bubbles are trapped in the gelation chamber	Use a pair of forceps to slowly lift one edge of the cell culture coverslip at a slight angle, until the air bubbles migrate to the edge of the coverslip and disperse, and then slowly place the cell culture coverslip back to rest on top of the two spacer stacks. Refill the chamber with gelation solution if necessary.
Step 40	Gel fails to form	Check the incubator and make sure the temperature is at 37°C. If the temperature is correct, the most likely next reason for failed gelation is bad reagents. Prepare new Stock X, APS, and TEMED solutions (ordering fresh reagents if needed) and redo the experiments.
Step 44	Gel remains attached to the coverslip or the Parafilm after ProK digestion	If the gel remains attached to the coverslip or Parafilm, it is most likely that the digestion step failed. Check the pH of the digestion buffer to make sure that it is ∼8.0. The ProK enzyme must be stored at −20°C. The gelled sample must be fully immersed in the ProK‐containing digestion buffer during the overnight digestion in step 43. If the above instructions were strictly followed, but the problem still persists, purchase fresh reagents and enzymes to make new ProK‐containing digestion solution.
	Gel cannot be located or is lost during removal of the digestion buffer	At this step, the gel is still relatively small and difficult to locate. First, we recommend illuminating the petri dish from different angles using a flashlight or a lamp to locate the gel. The gel will slightly scatter incident light and so should be visible when illuminated from different angles. Second, we recommend dispensing the aspirated digestion solution into another clean petri dish, rather than discarding it right away, so that if the gel is accidently drawn into the transfer pipet, it can possibly be recovered from the dispensed digestion solution. After the gel is secured, then discard the aspirated digestion solution appropriately.
Steps 44‐53	Gel is damaged	Always use a wet paint brush to handle the gel if needed. Be slow and careful when handling the gel. The most likely cause of gel damage is aspiration or solution addition in steps 46, 47, 48, and 51. During these steps, first locate the gel using the instructions listed above, and then slowly aspirate solution from around the gel, avoiding touching the gel itself. Tilt the petri dish to move liquids to the side of the dish opposite to the gel, so as to avoid potential contact with the gel when aspirating liquids. If only a small part of the gel is damaged, use a clean razor blade to cut off the damaged part, provided that the remaining part is big enough for downstream processing and imaging.
Steps 46‐48 and 51	Gel cannot be located	As the gel expands, it will be easier to locate. We recommend illuminating the petri dish from different angles using a flashlight or lamp to locate the gel. The gel will slightly scatter incident light and so should be visible when illuminated from different angles. Slightly tilt the petri dish at different angles to help with this process, as needed.
Step 53	Air bubbles are trapped between the bottom of the gel and the glass bottom of the 6‐well plate	Use a wetted paintbrush to gently press the top of the gel to expel large air bubbles. In the case of small air bubbles that were not removed, image regions of the sample that do not include these air bubbles during microscopy. Mounting smaller gels (e.g., ∼10 mm × 10 mm, which is still sufficiently large for imaging) is less likely to introduce air bubbles.
Other	Can I use this protocol for other types of specimens, for example, tissues?	No, this protocol is designed and optimized for visualizing immunostained microtubules in expanded HeLa cells. Please refer to other, more flexible and comprehensive protocols (Asano et al., 2018, and protocols posted at http://expansionmicroscopy.org) for imaging of other types of specimens.
	Can I store the expanded gel before mounting?	Yes, the expanded gel can be stored in water in the petri dish for up to 1 week at 4°C, in the dark to avoid photobleaching. For long‐term storage, remove the water from the petri dish containing the expanded gel, add 10 ml PBS solution, and store the petri dish for up to a few months at 4°C. The gel will shrink in PBS and can be re‐expanded, mounted, and imaged by first removing the PBS solution and then following steps 46‐54 of the protocol.
	Can I store the expanded and mounted gel?	Yes, the expanded and mounted gel can be stored for up to 1 week at 4°C in the dark.
	How should I properly dispose of used chemicals, gels, and solutions?	Follow instructions on the Material Safety Data Sheet (MSDS) provided by the manufacturers. Consult the Environment, Health and Safety (EHS) office of your institution for questions related to chemical and biological waste disposal.