Table 2 |.
Troubleshooting table
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 70 | Inefficient conjugation of oligonucleotide to antibody | Improper antibody starting amount | Measure the concentration of the starting antibody stock by A280 absorbance. Ensure that the conjugation amount is 100 μg |
| Carrier proteins within antibody stocks | Check the manufacturer specifications for carrier proteins (e.g., BSA, tissue culture supernatant, ascites or other protein stabilizer). Obtain carrier-free antibodies or purify the antibody before conjugation | ||
| Over-reduction of antibody by TCEP | Ensure that antibody conjugation is performed at room temperature (i.e., avoid incubation next to heatgenerating instruments) and that reduction time does not exceed 30 min | ||
| Improper staining result after conjugation to newly activated oligonucleotide | Reduction in maleimide functional groups from oligonucleotide | Ensure the the oligonucleotide is working by conjugating it to a known working antibody (e.g., aCD3). If not, then the deprotection procedure may have gone wrong, or maleimide groups have destabilized, in which case replace it with fresh stock | |
| Unsuccessful staining (e.g., no signal or nuclear staining) | Incompatible oligonucleotide-antibody pairing | If antibody staining works with conventional immunohistochemistry, repeat conjugation with a different oligonucleotide | |
| High fluorescence background for all channels and markers | Poor tissue morphology | Select samples that were frozen/fixed immediately after removing them from the subject. Confirm proper morphology with H&E staining | |
| The coverslip was contaminated | Ensure that the coverslip was not exposed to any fluorescent material (e.g., permanent marker during ethanol/methanol steps) and that all salt precipitates are removed before mounting the coverslip to the stage. Also ensure that paraffin was completely removed during baking and xylene washing | ||
| Weak antibody signal/high background | Low-abundance target antigen | Use ATTO550 or Alexa647 channels (Alexa488 has a higher background, especially in FFPE tissue). If this is insufficient, select a corresponding fluorescent oligonucleotide that is labeled with ATTO550 or Alexa647 (lower tissue autofluorescence) on both the 5′ and 3′ ends. If that does not work, consider trying a different antibody clone | |
| The antibody has not been titrated | Consider increasing the antibody staining concentration to increase the signal | ||
| Proper antibody staining but high background (for one channel) | Antibody staining at too high a concentration | Repeat staining at a two- to fourfold lower dilution | |
| Nonspecific fluorescent globules observed | The antibody aggregates | Spin down the antibody for 8 min at 12,000g at 4 °C to pellet aggregates (we recommend doing this monthly for the entire antibody panel). Extract antibody by pipetting from the top of the supernatant | |
| 133 | High fluorescence background for all channels and markers | Nonspecific oligonucleotide fluorescent signal | Make sure to perform a stripping cycle with stripping buffer before multicycle imaging to remove excess, nonspecific oligonucleotides |
| 143 | The vortex cluster is composed of two distinct clusters | Clustering is driven by the strength of a unifying marker (e.g., GATA3 stains basal epithelium and Th2 cells) | Over cluster (i.e., beyond the elbow point) and manually merge clusters |