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Storage: Store all kit components at -20°C. Stability: All components in this kit are stable for at least 12 months when stored at the recommended temperature. Do not exceed 5 freeze/thaw cycles. Application: The SignalStar kits are intended for fluorescent multiplex immunohistochemistry. Slide Number: This kit contains sufficient materials for the staining of 10 slides. |
SignalStar™ multiplex Immunohistochemistry (mIHC) is a technology that employs antibodies, oligonucleotides (oligos), and fluorophores to interrogate the cellular presence, location, function, and biomarker co-expression patterns. SignalStar technology enables the detection of multiple phenotypic and functional targets while maintaining spatial context and tissue architecture. These insights are essential for understanding how cells organize and interact to influence the tissue microenvironment and drive disease progression and response to therapy.
The power of the SignalStar system lies in the design of the SignalStar antibodies. These antibodies have been rigorously validated for use in formalin-fixed, paraffin-embedded (FFPE) tissues, and subsequently conjugated to unique oligo tags using site-specific conjugation and thorough purification methodologies. Using a highly specific network of complementary oligos and fluorophores, scientists can amplify the signal for 3-8 targets, even if they are in low abundance.
Figure 1. All antibodies in your plex size of choice (3-8 maximum unique oligo-conjugated antibodies) are added in cocktail in one primary incubation step. Complementary oligos with fluorescent dyes (channels: 488, 594, 647, and 750) amplify the signal of up to 4 oligo-conjugated antibodies in the first round of imaging by building oligo-fluorophore constructs attached to the antibody. If the plex size is greater than 4, the first round of oligos and fluorophores are gently removed, and a second round of amplification is performed to visualize up to 4 additional oligo-conjugated antibodies; the complementary oligo system and the use of the fluorophore removal process enables a second round of antibodies to be amplified from the same substrate, without cross-reactivity. The 2 images are then aligned and fused computationally with either proprietary or open-source software to generate an image consisting of up to 8 targets.
| Materials Included in SignalStar™ Secondary Antibody Kit |
| Up to 3 SignalStar oligo-conjugated secondary antibodies (see below) |
| Up to 3 SignalStar complementary oligos (see below) |
| Up to 3 Secondary blocking isotype control antibody |
| SignalStar™ Antibody Diluent A |
| SignalStar™ Antibody Diluent B |
| Materials Included in SignalStar™ Multiplex IHC Buffer Kit |
| Up to 7 SignalStar oligo-conjugated antibodies (see below) |
| Up to 7 SignalStar complementary oligos (see below) |
| SignalStar™ Antibody Diluent A |
| SignalStar™ Antibody Diluent B |
| SignalStar™ Amplification Buffer A |
| SignalStar™ Amplification Buffer B |
| SignalStar™ Amplification Oligo Set A: |
| 488 |
| 594 |
| 647 |
| 750 |
| SignalStar™ Amplification Oligo Set B: |
| 488 |
| 594 |
| 647 |
| 750 |
| SignalStar™ Ligation Buffer |
| T4 DNA Ligase (5 U/µL) |
| ATP (100 mM) |
| 10X dsDNase Buffer |
| dsDNase |
Included are SignalStar oligo-conjugated antibodies (A) and SignalStar complementary oligos (B) selected at the time of order and provided in sleeves with their respective oligo-antibody pair (C). See example below.
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PLEASE READ SOLUTION PREPARATION AND PROTOCOL IN ITS ENTIRETY PRIOR TO SETTING UP YOUR EXPERIMENT. |
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DO NOT COMBINE COMPLEMENTARY OLIGOS OF THE SAME FLUORESCENT CHANNEL. Imaging rounds can contain only 1 complementary oligo for each fluorescent channel. DO NOT COMBINE COMPLEMENTARY OLIGOS SPECIFIC TO THE SAME FLUORESCENT CHANNEL IN THE SAME IMAGING ROUND, AS IT WILL RENDER THE ASSAY RESULTS UNINTERPRETABLE. |
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DO NOT COMBINE ANTIBODIES FROM DIFFERENT PANELS. If you are running multiple panels simultaneously, a separate antibody/complementary oligo mix must be generated for each unique panel. |
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SOME SIGNALSTAR KIT COMPONENTS ARE VISCOUS. FLUORESCENT SIGNAL MAY BE VARIABLE OR DIMINISHED IF SOLUTIONS ARE NOT ACCURATELY MEASURED OR SUFFICIENTLY MIXED. Combine SignalStar kit components in 15 mL conical tubes using low retention pipette tips. Pipette slowly to ensure accuracy. Rotate end-over-end for 20 min at room temperature. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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Please confirm whether your SignalStar panel design requires 2 rounds of imaging. Utilize the Example SignalStar Panel Design and SignalStar Panel Design Worksheet in section 9.1 and 8.1 of this document for guidance and assistance. |
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Slides should be imaged within 8 hr of staining completion. Fluorescent signal may be diminished if slides are not imaged within this timeframe. |
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Confirm your microscope can detect the fluorophores provided in this kit. When imaging, there are 4 fluorescent channels in addition to DAPI that need to be acquired. Contact your instrument provider to confirm instrument settings. |
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It is recommended that a spectral library be created by imaging single stained slides for the spectra described above. This can enable better channel separation to help minimize the possibility of spectral bleed-through. |
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Usage of a DAPI concentrate is recommended rather than a mount that contains DAPI. Bright DAPI staining facilitates better image alignment. |
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Results are not guaranteed if there is any deviation from this protocol. The SignalStar protocol was developed and optimized with the designated antigen retrieval and staining steps. |
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The usage of a positive control slide is recommended. A tissue upon which chromogenic staining has confirmed the presence of all targets in the multiplex panel should be included in each run. |
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Tissue preparation and fixation methods may vary, therefore enough antibody reagent is supplied for use at a range of 1:50 to 1:200 dilution. It recommended that each antibody be used at 1:100 for initial testing to determine whether titration is required. |
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Drain off the incubation solutions and dH2O as much as possible throughout the staining process without allowing slides to dry out. Thoroughly flick liquid off from every slide after each step before continuing to the next. |
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The following SignalStar kit components can be thawed overnight at 4C prior to use:
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EACH SOLUTION SHOULD BE CREATED FRESH AND USED PROMPTLY. Please read SignalStar Imaging Round 1: Protocol for Use in section 5 in its entirety prior to creating solutions. |
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SignalStar kit components should be thawed at room temperature immediately prior to use unless otherwise indicated, and then stored on ice while in use. |
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Primary antibody diluent and primary antibodies are NOT included in this kit. Please use primary antibodies at recommended dilutions provided by the manufacturer in the recommended diluent. Confirm your final volumes match what is listed in the table below. |
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When purchasing your primary antibody, please make sure your primary antibody is validated for use with formalin-fixed, paraffin embedded tissues. You can see a comprehensive catalog of IHC-P-validated antibodies at www.cellsignal.com. |
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IF USING MORE THAN ONE PRIMARY AND SECONDARY ANTIBODY: Combine all primary antibodies from unique source species (ex. Rabbit mAb, Mouse mAb, and Rat mAb). DO NOT COMBINE PRIMARY ANTIBODIES FROM THE SAME SOURCE SPECIES. |
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SIGNALSTAR ANTIBODY DILUENTS ARE VISCOUS. FLUORESCENT SIGNAL MAY BE VARIABLE OR DIMINISHED IF SOLUTIONS ARE NOT ACCURATELY MEASURED OR SUFFICIENTLY MIXED. Combine SignalStar kit components in 15 mL conical tubes using low retention pipette tips. Pipette slowly to ensure accuracy. Rotate end-over-end for 20 min at room temperature. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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IF USING MORE THAN ONE PRIMARY AND SECONDARY ANTIBODY: Combine all secondary antibodies and complementary oligos used to detect unique source species (ex. Anti-rabbit, Anti-mouse, and Anti-rat).. |
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SIGNALSTAR ANTIBODY DILUENTS ARE VISCOUS. FLUORESCENT SIGNAL MAY BE VARIABLE OR DIMINISHED IF SOLUTIONS ARE NOT ACCURATELY MEASURED OR SUFFICIENTLY MIXED. Combine SignalStar kit components in 15 mL conical tubes using low retention pipette tips. Pipette slowly to ensure accuracy. Rotate end-over-end for 20 min at room temperature. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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IF USING MORE THAN ONE PRIMARY AND SECONDARY ANTIBODY: Combine all secondary blocking isotype control antibodies for included unique source species (ex. Rabbit mAb, Mouse mAb, and Rat mAb).. |
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Once prepared, the SignalStar Imaging Round 1 Solution should contain ALL antibodies (up to 7) ordered with your kit (including those for Imaging Round 1 and Imaging Round 2) and the complementary oligos for Imaging Round 1. Utilize the Example SignalStar Panel Design and SignalStar Panel Design Worksheet in section 9.1 and 8.1 of this document for guidance and assistance. |
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DO NOT COMBINE COMPLEMENTARY OLIGOS OF THE SAME FLUORESCENT CHANNEL. Do not combine complementary oligos from Imaging Round 1 and Imaging Round 2. |
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SIGNALSTAR ANTIBODY DILUENTS ARE VISCOUS. FLUORESCENT SIGNAL MAY BE VARIABLE OR DIMINISHED IF SOLUTIONS ARE NOT ACCURATELY MEASURED OR SUFFICIENTLY MIXED. Combine SignalStar kit components in 15 mL conical tubes using low retention pipette tips. Pipette slowly to ensure accuracy. Rotate end-over-end for 20 min at room temperature. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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SIGNALSTAR AMPLIFICATION BUFFERS ARE VISCOUS. FLUORESCENT SIGNAL MAY BE VARIABLE OR DIMINISHED IF SOLUTIONS ARE NOT ACCURATELY MEASURED OR SUFFICIENTLY MIXED. Combine SignalStar kit components in 15 mL conical tubes using low retention pipette tips. Pipette slowly to ensure accuracy. Rotate end-over-end for 20 min at room temperature. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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SIGNALSTAR AMPLIFICATION BUFFERS ARE VISCOUS. FLUORESCENT SIGNAL MAY BE VARIABLE OR DIMINISHED IF SOLUTIONS ARE NOT ACCURATELY MEASURED OR SUFFICIENTLY MIXED. Combine SignalStar kit components in 15 mL conical tubes using low retention pipette tips. Pipette slowly to ensure accuracy. Rotate end-over-end for 20 min at room temperature. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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EACH SOLUTION SHOULD BE CREATED FRESH AND USED PROMPTLY. Please read SignalStar Imaging Round 1: Protocol for Use in section 5 in its entirety prior to creating solutions. |
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Slide baking can be performed the day before you begin your experiment. This step allows for the paraffin wax to melt and the tissue better adhere to the slide. |
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1. Incubate slides for at least 30 min at 60°C. |
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Do not let slides dry out at any point once they are deparaffinized. Use a humidified chamber for all incubation steps. Make sure each solution covers the entirety of the tissue. |
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2. Incubate sections in 3 washes of xylene for 5 min each. |
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3. Incubate sections in 2 washes of 100% ethanol for 10 min each. |
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4. Incubate sections in 2 washes of 95% ethanol for 10 min each. |
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5. Wash sections 2 times in dH2O for 5 min each. |
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EDTA antigen retrieval in a pressure cooker is recommended to maximize retrieval of epitopes. This protocol describes the conditions that are recommended for the Biocare Medical Decloaking Chamber #DC2012. Device-specific settings. Manufacturer recommended operating instructions should be utilized for other pressure cookers. |
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7. Place 500 mL dH2O into the pressure cooker. |
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8. Place the slide holder into the pressure cooker, touching the heat shield. Partially cover with a slide container lid. |
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It may be advantageous to place a second 24-slide holder filled with 250 mL water and blank slides into the pressure cooker, and partially cover with a lid. |
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9. Seal the chamber and proceed with retrieval. Settings for the Biocare Medical Decloaking Chamber #DC2012 are 110°C for 30 min. |
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10. Carefully vent the device, then remove the lid. |
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11. Remove the slide container from the decloaking chamber and allow to cool on the bench top for 10 min. |
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13. Thoroughly flick off liquid from slides and incubate in 150 µL Primary Antibody Solution according to manufacturer's protocol. |
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Slides can be incubated overnight in the Primary Antibody Solution at 4°C in order to break up the protocol into multiple days. |
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14. Thoroughly flick off liquid from slides and immerse in 1X TBST for 30 sec. |
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15. Thoroughly flick off liquid from the slide and incubate in 150 µL Secondary Antibody Solution for 40 min at room temperature. |
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16. Thoroughly flick off liquid from slides and immerse in 1X TBST for 30 sec. |
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17. Thoroughly flick off liquid from slide and incubate in 150 µL Secondary Blocking Solution for 40 min at room temperature. |
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18. Thoroughly flick off liquid from slides and immerse in 1X TBST for 30 sec. |
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19. Incubate slides in 10% Neutral Buffered Formalin for 5 min at room temperature. |
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20. Thoroughly flick off liquid from slides and immerse in dH2O for 30 sec. |
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21. Incubate slides in 150 µL of SignalStar Imaging Round 1 Solution for 40 min at room temperature. |
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Slides can be incubated overnight at 4°C in order to break up the protocol into multiple days. |
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22. Thoroughly flick off liquid from slides and immerse in 1X TBST for 30 sec. |
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23. Incubate slides in 10% Neutral Buffered Formalin for 5 min at room temperature. |
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24. Thoroughly flick off liquid from slides and immerse in dH2O for 30 sec. |
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26. Incubate slides in 150 µL of SignalStar Ligation Buffer for 20 min at room temperature. |
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27. Thoroughly flick off liquid from slides and immerse in dH2O for 30 sec. |
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28. Prepare DAPI #4083 solution according to datasheet instructions. |
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29. Immerse slides in 1X TBST for 30 sec. |
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30. Counterstain with DAPI solution. |
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31. Immerse slides in 1X TBST for 30 sec. |
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32. Mount slides with ProLong Gold Antifade Reagent #9071. |
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33. Image slides as soon as possible. Signal should remain robust for up to 8 hr. |
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Once imaging is complete, fluorescent signal can be removed from slides so that another round of imaging can be performed. Perform Imaging Round 2 as soon as possible following the first round of imaging. |
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EACH SOLUTION SHOULD BE CREATED FRESH AND USED PROMPTLY. Please read SignalStar Imaging Round 2: Protocol for Use in section 7 in its entirety prior to creating solutions. |
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SIGNALSTAR IMAGING ROUND 2 IS ONLY NEEDED FOR ANTIBODY PANELS THAT REQUIRE TWO IMAGING ROUNDS. Utilize the Example SignalStar Panel Design and SignalStar Panel Design Worksheet in section 9.1 and 8.1 of this document for guidance and assistance. |
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SignalStar kit components should be thawed at room temperature immediately prior to use unless otherwise indicated, and then stored on ice while in use. |
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Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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Once prepared, the SignalStar Imaging Round 2 Solution should contain only the complementary oligos for Imaging Round 2. Utilize the Example SignalStar Panel Design and SignalStar Panel Design Worksheet in section 9.1 and 8.1 of this document for guidance and assistance. |
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NO SIGNALSTAR CONJUGATES ARE ADDED TO THE IMAGING ROUND 2 SOLUTION. |
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DO NOT COMBINE COMPLEMENTARY OLIGOS OF THE SAME FLUORESCENT CHANNEL. Do not combine complementary oligos from Imaging Round 1 and Imaging Round 2. |
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SIGNALSTAR ANTIBODY DILUENTS ARE VISCOUS. FLUORESCENT SIGNAL MAY BE VARIABLE OR DIMINISHED IF SOLUTIONS ARE NOT ACCURATELY MEASURED OR SUFFICIENTLY MIXED. Combine SignalStar kit components in 15 mL conical tubes using low retention pipette tips. Pipette slowly to ensure accuracy. Rotate end-over-end for 20 min at room temperature. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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SIGNALSTAR ANTIBODY DILUENTS ARE VISCOUS. FLUORESCENT SIGNAL MAY BE VARIABLE OR DIMINISHED IF SOLUTIONS ARE NOT ACCURATELY MEASURED OR SUFFICIENTLY MIXED. Combine SignalStar kit components in 15 mL conical tubes using low retention pipette tips. Pipette slowly to ensure accuracy. Rotate end-over-end for 20 min at room temperature. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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SIGNALSTAR ANTIBODY DILUENTS ARE VISCOUS. FLUORESCENT SIGNAL MAY BE VARIABLE OR DIMINISHED IF SOLUTIONS ARE NOT ACCURATELY MEASURED OR SUFFICIENTLY MIXED. Combine SignalStar kit components in 15 mL conical tubes using low retention pipette tips. Pipette slowly to ensure accuracy. Rotate end-over-end for 20 min at room temperature. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly. |
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EACH SOLUTION SHOULD BE CREATED FRESH AND USED PROMPTLY. Please read SignalStar Imaging Round 2: Protocol for Use in section 7 in its entirety prior to creating solutions. |
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SIGNALSTAR IMAGING ROUND 2 IS ONLY NEEDED FOR ANTIBODY PANELS THAT REQUIRE TWO IMAGING ROUNDS. Utilize the Example SignalStar Panel Design and SignalStar Panel Design Worksheet in section 9.1 and 8.1 of this document for guidance and assistance. |
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1. After image acquisition for Imaging Round 1, soak slides in dH2O for ≥30 min to gently remove coverslips without damaging tissue. |
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2. Incubate slides in 150 µL of Fluorescence Removal Solution for 2 hr at 37°C. |
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3. Immerse slides in dH2O for 30 sec. |
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4. Optional: |
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5. Incubate slides in 150 µL of SignalStar Imaging Round 2 Solution for 40 min at room temperature. |
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6. Thoroughly flick off liquid from slides and immerse in dH2O for 30 sec. |
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8. Incubate slides in 150 µL of SignalStar Ligation Buffer for 20 min at room temperature. |
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9. Thoroughly flick off liquid from slides and immerse in dH2O for 30 sec. |
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10. Prepare DAPI #4083 solution according to datasheet instructions. |
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11. Immerse slides in 1X TBST for 30 sec. |
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12. Counterstain with DAPI solution. |
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13. Immerse slides in 1X TBST for 30 sec. |
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14. Thoroughly flick off liquid from slides and immerse in dH2O for 30 sec. |
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15. Mount slides with ProLong Gold Antifade Reagent #9071. |
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16. Image slides as soon as possible. Signal should remain robust for up to 8 hr. |
| Oligo-Conjugated Antibody | Complementary Oligo | Product Pair # | Imaging Round | 488 | 594 | 647 | 750 |
(See Appendix 9.1 for an example panel design.)
| Amplification Round | Step # | ✓ | Step |
| Amplification Round 1 | 1 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 2 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 3 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 4 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 2 | 5 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 6 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 7 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 8 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 3 | 9 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 10 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 11 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 12 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 4 | 13 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 14 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 15 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 16 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 5 | 17 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 18 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 19 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 20 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 6 | 21 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 22 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 23 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 24 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 7 | 25 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 26 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 27 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 28 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 8 | 29 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 30 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 31 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 32 | ☐ | Immerse slides in dH2O for 30 sec. |
| Amplification Round | Step # | ✓ | Step |
| Amplification Round 1 | 1 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 2 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 3 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 4 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 2 | 5 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 6 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 7 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 8 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 3 | 9 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 10 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 11 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 12 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 4 | 13 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 14 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 15 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 16 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 5 | 17 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 18 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 19 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 20 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 6 | 21 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 22 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 23 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 24 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 7 | 25 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 26 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 27 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 28 | ☐ | Immerse slides in dH2O for 30 sec. | |
| Amplification Round 8 | 29 | ☐ | Incubate in Amplification Solution 1 for 8 min. |
| 30 | ☐ | Immerse slides in dH2O for 30 sec. | |
| 31 | ☐ | Incubate in Amplification Solution 2 for 8 min. | |
| 32 | ☐ | Immerse slides in dH2O for 30 sec. |
| Oligo-Conjugated Antibody | Complementary Oligo | Product Pair # | Imaging Round | 488 | 594 | 647 | 750 |
| Goat Anti-Rabbit IgG (H&L) Antibody | Complementary Oligo (CO-0055-488) |
30599 | 1 | ● | |||
| PD-L1 (E1L3N®) XP® Rabbit mAb (SignalStar™ Conjugate 0005) |
Complementary Oligo (CO-0005-594) |
28249 | 1 | ● | |||
| TIM-3 (D5D5R™) XP® Rabbit mAb (SignalStar™ Conjugate 0010) |
Complementary Oligo (CO-0010-647) |
15231 | 1 | ● | |||
| Ki-67 (8D5) Mouse mAb (SignalStar™ Conjugate 0014) |
Complementary Oligo (CO-0014-750) |
56398 | 1 | ● | |||
| CD8ɑ (D8A8Y) Rabbit mAb (SignalStar™ Conjugate 0004) |
Complementary Oligo (CO-0004-488) |
45747 | 2 | ● | |||
| CD68 (D4B9C) XP® Rabbit mAb (SignalStar™ Conjugate 0007) |
Complementary Oligo (CO-0007-594) |
77318 | 2 | ● | |||
| CD20 (E7B7T) XP® Rabbit mAb (SignalStar™ Conjugate 0011) |
Complementary Oligo (CO-0011-647) |
36775 | 2 | ● | |||
| Pan-Keratin (C11) Mouse mAb (SignalStar™ Conjugate 0003) |
Complementary Oligo (CO-0003-750) |
97227 | 2 | ● |
How are the SignalStar Multiplex IHC Kits & Reagents validated?
CST thoroughly validates each antibody available in the SignalStar Multiplex IHC Panel Builder menu. Various combinations of antibodies are tested through titration and fluorophore pairing, and in both rounds of imaging. Testing is performed on a variety of tumors and tissue types. We also rigorously test the parent antibodies used in the traditional chromogenic assay, as they serve as the foundation of this fluorescent assay. We are not able to test all multiplex configurations or tissues. Please contact customer support if you have any questions.
Do I need to optimize the SignalStar Multiplex IHC Kits & Reagents for the type of tissue I’m using?
The SignalStar Multiplex IHC Kits & Reagents have been optimized with respect to fluorophore pairing and order of antibodies. As tissues vary in quality and expression level of targets, increasing the concentration of antibodies in your panel by 2-fold or decreasing by 0.5 fold can help achieve optimal signal in your experiments. Small titrations such as this may be necessary in order to achieve lower levels of background and higher S/N levels.
What are the imaging instrument specifications required to image SignalStar Multiplex IHC?
There are four fluorescent channels in addition to DAPI that need to be acquired. Contact your instrument provider to confirm instrument specifications. Ensure the Texas Red filter set is used to visualize the 594 channel. The TRITC filter is not ideal and may result in diminished signal output.
| Fluorophore Channel | Excitation (nm) | Emission (nm) | Laser Line | Common Filter Set |
| 488 | 488 | 520 | 488 | FITC |
| 594 | 590 | 618 | 561/594 | Texas Red |
| 647 | 650 | 668 | 594/633 | Cy®5 |
| 750 | 752 | 776 | 633 | Cy®7 |
SignalStar mIHC is a signal amplification-based technology, therefore for initial imaging, we recommend auto-exposing control tissues to identify optimal exposure times for each channel.
For some instruments, spectral bleed-through can occur between the 594 and 647 channels. Decreasing the concentration of antibodies placed in the 647 channel may help with bleed-through. Spectral bleed-through can also be considered during panel design so that a strong phenotypic marker is spectrally separated from weaker expressing targets.
Does this assay work on frozen tissue?
SignalStar Multiplex IHC Kits & Reagents haven’t yet been validated for use in frozen tissues.
Do you have mouse-reactive antibodies available?
Yes, please select "Mouse" in the first step of the SignalStar Multiplex IHC Panel Builder to see our mouse-reactive menu.
I don't see my target of interest in your menu of available antibodies. Can I still use it in my panel in some way?
SignalStar Multiplex IHC Kits & Reagents haven't yet been validated for use with antibodies outside of our menu. We're in the process of developing custom solutions for using your own antibodies in the SignalStar Multiplex IHC assay.
SignalStar mIHC is a non-destructive technology. Therefore, in order to use your antibodies of interest, you may perform direct immunofluorescence on the same tissue after the SignalStar assay. The SignalStar™ Fluorescence Removal Kit #32722 enables you to remove the fluorescent oligos after SignalStar mIHC in order to stain and visualize direct immunofluorescence.
Can I combine antibodies used in this assay with direct conjugates?
SignalStar Multiplex IHC Kits & Reagents have been validated for use in combination with direct conjugates. The SignalStar™ Fluorescence Removal Kit #32722 enables you to remove the fluorescent oligos after SignalStar mIHC in order to stain and visualize direct immunofluorescence directly after SignalStar mIHC. We have found that many direct conjugates against strong cell surface markers work well when used in this manner. Please see our recent poster presented at AACR this past spring for more information. In addition, please see our line of directly conjugated antibodies for use in direct immunofluorescence imaging.
When comparing my SignalStar staining to the chromogenic staining on serial sections, I see more positive cells. How do I know if this excess staining is correct?
During the course of optimization, we've found that fluorescent staining may show higher %-positivity than chromogenic staining. To ensure any excess staining is specific, confirm that the correct subcellular localization and co-localization with other stains are demonstrated. For example, if all CD8+ cells are CD3+, any excess CD8+ staining compared to the chromogenic is most likely correct.
How long after the completion of staining can I wait to image my slides?
For Imaging Round 1, the staining should show robust signal when imaged up to 8 hr post completion of staining. For Imaging Round 2, imaging should be performed as close to the completion of staining as possible, but should remain robust for up to 8 hr.
Do I need to optimize the SignalStar Multiplex IHC Kits & Reagents for the type of tissue I'm using?
The SignalStar Multiplex IHC Kits & Reagents have been optimized with respect to fluorophore pairing and order of antibodies. As tissues vary in quality and expression level of targets, increasing the concentration of antibodies in your panel by 2-fold or decreasing by 0.5 fold can help achieve optimal signal in your experiments.
What is an appropriate positive control to include in this assay? Are multiple controls necessary?
Any tissue shown to be positive for each target via chromogenic IHC can serve as a positive control tissue. Each target will therefore require a positive control, which may sometimes necessitate multiple controls. For optimal comparison, the sections should be as close to serial as possible.
Can I perform alternative antigen retrieval methods than those provided in the protocol?
For optimal results, we do not recommend deviating from the SignalStar protocols. However, if you do not have access to the antigen retrieval method detailed in the protocol, you may try using a microwave, which has shown to result in reduced fluorescent signal. We do not recommend using a microwave when detecting low abundance targets, and we cannot guarantee your result.
To perform antigen retrieve using a microwave:
Prepare 1X EDTA Unmasking Solution: To prepare 250 mL of 1X EDTA unmasking solution, dilute 25 ml of SignalStain® EDTA Unmasking Solution (10X) (#14747) with 225 mL of dH2O. Heat slides in a microwave submersed in 1X EDTA unmasking solution until boiling is initiated (~2.5 minutes at power level 10). Follow with 15 min at a sub-boiling temperature (95°-98°C). In most common microwaves, this equates to 8 minutes at power level 3, then 7 minutes power level 2. Then remove from the microwave and place the container of slides under a dH2O faucet and add water directly to the slide container until all EDTA is replaced by dH2O. No cooling is necessary.
I am looking to run this assay on adipose tissue / brain / normal kidney which has a high level of autofluorescence. Do you have any example data that you can provide me to demonstrate that this assay will work in this tissue type?
While we utilize a wide variety of tumor and tissue types during the course of our optimization process, we cannot account for all tissues and expression levels. This assay should work in FFPE tissue 4-5 uM in thickness, assuming that our recommended protocol is followed. Furthermore, due to the high level of amplification that this assay provides, even very high autofluorescence levels may be overcome with the resulting strong, specific signal.
Can I use an alternative mounting reagent instead of ProLong Gold Antifade Reagent #9071?
Prolong Antifade mounting media are optimal for this protocol. Internal testing has demonstrated that SlowFade Antifade Reagents can negatively impact signal intensity.
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