TABLE 2.

Troubleshooting table.

Step	Problem	Possible reason and solution
1	Low number of cells in the pellets	Inadequate sample (cell suspension) collection. At least 3 million cells should be used for each pellet, but it is desirable to have more in case you lose cells or the pellet during processing. Make sure that the pellet can be visualized after fixation and before processing. Hypocellular suspensions may need pelleting, suspending and re-pelleting several times to make an appropriate pellet. Do not go ahead if you cannot see a good pellet. Re-collect sample with sufficient number of cells
3	Cells are spread in the agar pellet	Agar pellets were prepared too slowly or agar was not adequately heated. If cells are spread you will not be able to see them in adequate numbers on the electron microscope. Re-prepare another agar pellet and check if the pellet can be seen on the tube bottom (good pellet) and not spread in the tube wall (bad pellet)
4	Watery agar pellet (agar pellet dissolves when taken out from the tube)	Supernatant was not adequately taken out from the tube after centrifugation of the cell suspension (before adding liquid agar). Re-prepare another agar pellet
6, 7	Cells are poorly visible during cryostat microtomy	If the initial pellet was made with enough cells, this problem is probably due to inadequate freezing of the pellet (incorrect position). In our experience, pellets should be frozen intact and with the bottom area (in which the cells are more concentrated) upright. This will allow better visualization of the cells during cryostat microtomy and also will guarantee sufficient number of cells to be analyzed on the thin sections. It is preferable to have a small area containing high number of cells than a larger area with spread cells. In this case, you will see a low number of cells on the electron microscope. Re-prepare samples and freeze adequately
20	Excessive growing of gold particles and/or background staining (Fig. 4)	The time of incubation with silver enhancement components is not optimized and probably was too long. Room temperature is too high. Silver enhancement is time-dependent. The enhancement time is the time required to obtain an adequate amplification of the Nanogold particles. Reduce the incubation time and test different times to find which one works best for your antigen. Check the room temperature. At 16 °C the developer solution is stable (no self-nucleation occurs) for at least 20 min; at 20 °C, the solution is stable for at least 15 min, and at 24 °C for at least 10 min. After this time, background staining may be observed. We established 10 min at ~20 °C as an ideal time period for our immunolabeling experiments
29	Resin blocks are soft	Polymerization problems can occur owing to improper oven temperature setting, insufficient time of polymerization or oscillating oven temperature. It is very important to complete resin polymerization at a constant temperature of 60 °C. Extend the polymerization time for extra few hours at the same temperature. Other possible reasons include inadequate mixing of resin components or out-of-date accelerator. In these cases, sample processing should be repeated. Water condensation may also have contaminated the resin mixture. Keep the resin dry and do not open it until it reaches room temperature
32	Trouble in cutting high-quality ultrathin sections for TEM	Ultramicrotomy requires high level of expertise and many hours of practice. Several factors such as knife angle, cutting speed, block size and water meniscus level can interfere in the procedure. One solution is to enlist the assistance of an experienced ultramicrotomist
33	Precipitation of lead on the thin sections	Contaminated lead citrate solution or inadequate washing of the grids after staining. Re-prepare the lead citrate solution. Rinse grids adequately using double-distilled water
34	Poor labeling	Inadequate fixation or primary antibody concentration is not optimized. Start using just 4% (wt/vol) paraformaldehyde as a primary fixative. Test the primary antibody by using different concentrations. In our hands 1 h of incubation at room temperature is sufficient to label different antigens (Table 1) when the primary antibody concentration is adequate. Try a different antibody that has previously worked for immunocytochemistry studies
	Excessive labeling	Primary antibody concentration is not optimized (too high concentration). Test the primary antibody by using different dilutions