SignalStar Multiplex IHC kits and reagents are optimized and validated for both the manual protocol and BOND RX Fully Automated Research Stainer by Leica Biosystems protocol. However, if you do need to troubleshoot your assay, please see the list of potential issues, possible causes, and recommendations below.
| Description | Possible Causes | Recommendation |
|---|---|---|
| I see no fluorescent signal in any channel, my slide is blank. | It's 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's possible that the tissue didn’t express the biomarker of interest. |
It's necessary to use a control slide in order to rule out any potential reagent issues. Perform chromogenic staining to confirm biomarker expression in tissue. |
| Description | Possible Causes | Recommendation |
|---|---|---|
| I see signal in all channels, but it's much weaker than expected. | SignalStar reagents and solutions are viscous. It's 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 by flicking the slide 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 BOND RX 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's 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's 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. | |
| Incorrect fluorescent imager settings or filter sets were used when scanning. |
Confirm the correct filter set was used to visualize the 488, 594, 647, and 750 nm channels. Please see the fluorescent channel details provided in the protocol. Ensure the Texas Red filter set is used to visualize the 594 nm channel. The TRITC filter is not compatible. |
| 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 marker. | 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's 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. | |
| Incorrect fluorescent imaging settings or filter sets were used when scanning. | Confirm the correct filter set is used to visualize the 488, 594, 647, and 750 nm channels. Please see the fluorescent channel details provided in the protocol. Ensure the Texas Red filter set is used to visualize the 594 nm channel. The TRITC filter is not compatible. |
| 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 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're 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 the 488, 594, and 750 nm channels, but only 6 in the 647 nm channel: 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. | |
| I'm seeing quite a bit of background (that doesn't match the chromogenic) in the necrotic areas of my tissue section. Is there any way to decrease this? | Necrotic tissue can be a challenge to stain with any assay. It is often “stickier” than other parts of the tissue when it comes to non-specific binding of dyes, oligos and antibodies themselves. | You can try to titrate the antibody down such that the specific signal remains, but the non-specific signal in the necrotic area doesn't. Alternatively, if the staining in the necrotic areas is just as bright as the specific signal elsewhere, you can simply focus on imaging the non-necrotic areas if possible. |
| I'm working with a highly autofluorescent brain tissue section and seeing a lot of background in the 488 nm channel. What can I do to minimize this? | There may be high levels of autofluorescence combined with weak signal intensity of the detected biomarker. |
While we utilize a wide variety of tumor and tissue types during the course of our optimization, we have not performed validation on every type. Tissue that's highly autofluorescent, hasn't been fixed right away, etc., can be difficult to stain with any assay. You can try to titrate the oligo-conjugated antibody in an effort to increase or decrease the signal you're seeing. Additionally, reagents aimed at decreasing autofluorescence such as TrueBlack Liposfuscin can be used. Autofluorescence can be considered during panel design so that a strong phenotypic marker is placed in the 488 nm channel rather than weaker expressed biomarkers. |
| Description | Possible Causes | Recommendation |
|---|---|---|
| I see fluorescent signal from multiple biomarkers in a single channel. | It's possible that complementary oligos from Imaging Round 1 and Imaging Round 2 were incorrectly combined, amplifying 2 biomarkers 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. |
| Incorrect fluorescent imager settings or filter settings were used when scanning. |
Confirm the correct filter set is used to visualize the 488, 594, 647, and 750 nm channels. Please see fluorescent channel details provided in the protocol. Ensure the Texas Red filter set is used to visualize the 594 nm channel. The TRITC filter is not compatible. A spectral library can be used to computationally unmix fluorescent signals if the characteristics of your imaging instrument are not ideal to separate these fluorescent channels. |
|
| Similar signal from the biomarker is present in more than one fluorescent channel. For example, the staining in the 647 nm channel looks similar to the biomarker present in the 594 nm channel. |
This could be due to a lack of biomarker expression in the 594 nm channel such that there is little or no signal in this channel. Concomitantly, there may be strong expression in the 647 nm channel, such that spectral bleed-through results in signal in the 594 nm channel. Weak specific signal in the 594 nm channel may be drowned out due to this phenomenon. |
Decreasing the amount of antibody placed in the 647 nm channel may help with this bleed-through of signal. Spectral bleed-through can be considered during panel design so that a strong phenotypic marker is spectrally separated from weaker expressing markers. |
| 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. | The aspirating probe cleaning cycle was not run after performing the SignalStar assay on both Imaging Round 1 and Imaging Round 2. | ALWAYS run an aspirating probe cleaning cycle after every SignalStar 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 open containers are not overfilled with SignalStar reagents. Scan the container again. If it's listed as “Empty,” click “Mark not empty.” Click “Refill.” Ensure that the appropriate volume of reagent is added. Some reagents may require multiple containers, as indicated in the BOND RX protocol. Contact your instrument provider for additional information and assistance. |
| I received this error message in the BOND RX software: “There is insufficient reagent.” | 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 the 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. |
Visit the Technical Support page to search for SignalStar-related troubleshooting information and answers to technical questions.
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U.S. Patent No. 10,781,477, foreign equivalents, and child patents deriving therefrom.
