SignalStar Multiplex IHC Kits & Reagents are optimized and validated for both the manual protocol and BOND RX Fully Automated Research Stainer by Leica Biosystems protocol. However, if you need to troubleshoot your assay, please see the list below for potential issues, possible causes, and recommendations.
| Description | Possible Causes | Recommendation |
|---|---|---|
| I see no fluorescent signal in any channel, my slide is blank. | It is possible that a critical component of the kit was left out during the staining process. | Confirm all reagents were added prior to performing the assay. |
| It is possible that the tissue didn’t express the target of interest. | It is necessary to use a control slide in order to rule out any potential reagent issues. |
| Description | Possible Causes | Recommendation |
|---|---|---|
| I see signal in all channels, but it is much weaker than expected. | SignalStar reagents and solutions are viscous. It is possible there was insufficient mixing of the SignalStar solutions, particularly Amplification Solution 1 and Amplification Solution 2. | Combine all SignalStar kit components using low retention pipette tips and rotate end-over-end for 20 min at room temperature. |
| Residual Amplification Solution 1 may have remained after the wash steps prior to applying Amplification Solution 2. | Ensure that each slide is completely immersed in dH2O and that the excess liquid is removed from the surface of the tissue via flicking prior to applying the amplification solution. | |
| Insufficient washing of solutions on the BOND RX autostainer. | Ensure all wash steps are selected as “Open” in the BOND RX software as outlined in the protocol. | |
| Fluorescent signal may be diminished if slides are not imaged within 8 hr of staining. | Ensure that slides are imaged as soon as possible after staining, within 8 hr max. | |
| It is possible that fewer than 8 complete rounds of amplification were performed during the manual protocol. | Use the checklists provided in the manual protocol during these steps. | |
| It is possible that fewer than 6 complete rounds of amplification were performed during the BOND RX protocol. | Use the checklists provided in the BOND RX protocol during these steps. | |
| While all antibodies are thoroughly validated for use in many tissues, tissue quality and expression levels vary. | Increasing the amount of antibody (2-fold) will increase signal intensity. |
| Description | Possible Causes | Recommendation |
|---|---|---|
| I see no signal in one of the fluorescent channels, but chromogenic staining on a serial section demonstrated strong signal for this target. | Complementary oligo was not added for the fluorescent channel. | Confirm all reagents were added prior to performing the assay. |
| Amplification oligos A or B were not added for the fluorescent channel. | Confirm all reagents were added prior to performing the assay. | |
| It is possible that the incorrect laser and filter set were used to image the slide. | The protocols contain information about the lasers, filter sets, excitations, and emissions of all of the fluorophores used in this assay. Confirm your instrument settings match. |
| Description | Possible Causes | Recommendation |
|---|---|---|
| There is background fluorescence in certain channels of my panel. | While all antibodies are thoroughly validated for use in many tissues, tissue quality and expression levels vary. | Decreasing the amount of antibody (0.5-fold) may help to decrease background levels while maintaining target signal intensity. |
| All antibodies have been validated in the context of the antigen retrieval methodology described in the protocol. If an alternative method is used, it may result in higher background levels. | Ensure you are using the antigen retrieval method outlined in the protocol. | |
| Using the manual protocol, fewer amplification cycles can be performed in the channels to reduce noise. | For example, to perform 8 amplification cycles in channel 488, 594, and 750, but only 6 in 647: make amplification solutions for all channels for 6 amplification rounds. Then, make separate amplification solutions consisting of amplification oligos for 488, 594, and 750 to be used for the last 2 amplification rounds. |
| Description | Possible Causes | Recommendation |
|---|---|---|
| Similar signal from the target is present in more than one fluorescent channel; for example, the staining in the 647 channel looks similar to the target present in the 594 channel. | This could be due to a lack of target expression in the 594 channel such that there is little/no signal in this channel. Concomitantly, there may be strong expression in the 647 channel, such that the spectral bleed-through results in signal in the 594 channel. Weak specific signal in the 594 channel may be drowned out due to this phenomenon. | Decreasing the amount of antibody placed in the 647 channel may help with this bleed-through of signal. Spectral bleed-through can be considered during panel design so a strong phenotypic marker is spectrally separated from weaker expressing targets. |
| Description | Possible Causes | Recommendation |
|---|---|---|
| I see fluorescent signal from multiple targets in a single channel. | It is possible that complementary oligos from Imaging Round 1 and Imaging Round 2 were incorrectly combined, amplifying 2 targets within the same channel. | Ensure that you do not combine complementary oligos of the same fluorescent channel in the same imaging round. Imaging rounds can contain only 1 complementary oligo for each fluorescent channel. |
| Description | Possible Causes | Recommendation |
|---|---|---|
| The BOND titration insert has lifted out of its carrier causing the BOND RX autostainer to malfunction and not complete the staining run. | Aspirating probe cleaning cycle was not run after performing SignalStar assay on both Imaging Round 1 and Imaging Round 2. | ALWAYS run an aspirating probe cleaning cycle after every SignalStar slide run. Contact your instrument provider for additional information and assistance. |
| I received an “Empty” container error message on the BOND RX software. | The BOND RX autostainer will consider containers to be “Empty” if they are overfilled. | Ensure that the open containers are not overfilled with SignalStar reagents. Some reagents may require multiple containers, as indicated in the protocol. Contact your instrument provider for additional information and assistance. |
| I received an error message stating, “There is insufficient reagent” on the BOND RX software. | The BOND RX autostainer may consider reagents to be "insufficient" despite using the correct volume of solution indicated in the SignalStar Multiplex IHC protocol. | Repeat attempt to fill and scan the container. You may need to use a new container to alleviate this error. Contact your instrument provider for additional information and assistance. |
