Table 3 |.
Troubleshooting table
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 2 | No bubbles in fluorophore bleaching solution | Incorrect lighting source or bleaching solution used | Use white LED light for fluorophore inactivation; use freshly made bleaching solution |
| 10 | Blurry images | The slide is not flat on microscope stage or there are bubbles between the slide and the coverslip | Examine the slide holder for precipitate and remove it; ensure slides are loaded properly in the slide holder; re-coverslip; adjust the focus points |
| 18 | Weak signal | Low signal can occur because of low-level antigen expression. Direct immunofluorescence using conjugated antibodies does not provide the signal amplification that can be generated in indirect immunofluorescence | Increase the exposure time while acquiring the image; increase the antibody concentration during the staining step; use the corresponding unconjugated antibodies in the first round instead of the conjugated antibody to see if signal amplification from indirect immunofluorescence improves signal; if necessary, find an alternative antibody |
| Saturated signal | Abundant antigen in sample or excessive amount of antibody. Exposure time too long | Decrease the antibody concentration used during the staining step; decrease the incubation time of the sample with antibody; decrease the exposure time during image acquisition | |
| 31 | Cell loss | Difficult tissue type (very low cell density). Insufficient tissue fixation. Damage from t-CyCIF procedure (e.g., rough handling of samples during washing) | Check pre-analytical variables. Be cautious in handling of samples during application of antibodies and washing steps, as well as during manipulation of coverslips |
| Signal present after fluorophore bleaching step | Insufficient fluorophore inactivation | Avoid Alexa Fluor 546-, Alexa Fluor 568-, and Alexa Fluor 594-conjugated antibodies because they are difficult to inactivate; dilute conjugated antibodies further; extend fluorophore bleaching time; check that light is hitting the sample | |
| Box 3, step 9 | Leica Bond RX machine fails to start | Incorrect setup of machine | Read the manufacturer’s instructions; check the protocol and reagent setup carefully; contact vendor |