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SignalStar™ Multiplex Immunohistochemistry Assay for Use on BOND RX Fully Automated Research Stainer by Leica Biosystems

Product Information:

signalstar-8plex  

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 Symbols

Contents

1 Introduction: The SignalStar™ Staining Methodology

SignalStar multiplex immunohistochemistry (mIHC) is a tool 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.

SignalStar Multiplex IHC WorkFlow Diagram

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.

2 Solutions and Reagents

2.1 Included Kit Components

Materials Included in Kit
Up to 8 SignalStar oligo-conjugated antibodies (see below)
Up to 8 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 up to 8 SignalStar oligo-conjugated antibodies (A) and up to 8 SignalStar complementary oligos (B) selected at the time of order and provided in sleeves with their respective oligo-antibody pair (C). See example below.

SignalStar Oligo Antibodies

2.2 Required Reagents Not Included

  • Tris Buffered Saline with Tween 20 (TBST-10X) #9997
  • DAPI #4083
  • ProLong Gold Antifade Reagent #9071
  • Nuclease-free Water #12931
  • 10% Neutral Buffered Formalin
  • Low Retention Pipette Tips

2.3 Required Reagents Not Included - Available from Leica Biosystems

  • BOND Research Detection System #DS9455
  • BOND Aspirating Probe Cleaning Kit #CS9100
  • BOND Titration Kit (10 containers, 50 inserts) #OPT9049
  • BOND Open Containers 30 mL (10 pack) #OP309700
  • BOND Open Containers 7 mL (10 pack) #OP79193
  • BOND Universal Covertiles (160 pack) #S21.4611
  • BOND TM Epitope Retrieval 2-1 L (RTU) #AR9640
  • BOND Dewax Solution 1 L (RTU) #AR9222
  • BOND Wash Solution 10X Concentrate, 1L #AR9590

3 Important Considerations Before You Begin

Warning

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.
 
Warning

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.
Caution

Use the BOND RX software to set up protocols and reagents PRIOR to creating your solutions. Create containers, protocols, and a new BOND Research Detection System. A BOND Research Detection System is required to run this protocol.
Caution

After performing each round of the SignalStar assay, always run an aspirating probe cleaning on the BOND RX.
Caution

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 10.1 and 9.1 of this document for guidance and assistance.
Caution

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.
 
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
Note

It is recommended that a spectral library be created by imaging single stained slides for the spectra described above. This will enable better unmixing to help minimize the possibility of spectral bleed-through.
Note

Usage of a DAPI concentrate is recommended rather than a mount that contains DAPI. Bright DAPI staining facilitates better image alignment.
Caution

Fluorescent signal may be variable or diminished if solutions are not sufficiently mixed. Prior to usage, spin down each reagent at 1,000 rpm for 30 sec, then slowly mix by pipetting. When using reagents, pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when not in use. SignalStar solutions should be used promptly once all reagents have been combined for the run.
Caution

Optimally, slides should be imaged within 8 hr of staining completion. Fluorescent signal may be diminished if slides are not imaged within this timeframe.
Note

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.
Note

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.
Note

It is recommended that each antibody be used at 1:100. However, enough antibody reagent is supplied for usage at either 1:50 or 1:200 dilutions.

4 BOND RX Autostainer Setup

Caution

Use the BOND RX software to set up protocols and reagents PRIOR to creating your solutions. Requires BOND RX software version 7.0 or greater.

4.1 New Reagent Creation on BOND RX Autostainer

The following reagents must be created on the BOND RX software for the SignalStar assay:

BOND Titration Container

 

1. MARKER (= SignalStar Staining Solution)
 

2. SignalStar Amplification Solution 1
 

3. SignalStar Amplification Solution 2
 

4. SignalStar Ligation Solution
 

5. SignalStar Fluorescent Removal Solution

BOND 7 mL Open Container

 

6. 10% Neutral Buffered Formalin
Caution

Select “Hazardous” when creating 10% Neutral Buffered Formalin reagent to ensure the waste is disposed of properly.

BOND 30 mL Open Container

 

7. 0.1X TBST
 

8. 1X TBST (Required for linking to BOND Research Detection System)

4.2 BOND Research Detection System Setup

 

1. Create a new BOND Research Detection System named "CST SignalStar."
 

2. Link 30 mL open container with 1X TBST to the CST SignalStar BOND Research Detection System.
Caution

The open container in Step 2 must be named distinctly (e.g., ‘1X TBST' instead of '0.1X TBST').

4.3 Create BOND RX Protocols for SignalStar Imaging Round 1 and Imaging Round 2

 

1. In the BOND RX software, select the "Protocol Setup" tab.
 

2. Select "*IF Protocol" (ensure that Leica is listed in the "Modified by" column).
 

3. Copy protocol and rename it "CST SignalStar Imaging Round 1." Add the abbreviated name "CST Rd1."
 

4. Select "Show wash steps."
 
5. 
 		 Delete all steps and add the steps found in section 9.2 of this document entitled "BOND RX Protocol Setup Checklist: Imaging Round 1."

 
 

6. Select "CST SignalStar" as preferred Detection System.
 

7. Select "Create Protocol."
 

8. Click Save and click Yes to acknowledge the caution message.
 

9. Create a copy of "CST SignalStar Imaging Round 1" protocol.
 

10. Change the name of the copy to "CST SignalStar Imaging Round 2" with the abbreviated name "CST Rd2."
 

11. Select "Show wash steps."
 
12. 
 		 Delete Steps 1-11 and add steps 1-10 as listed in section 9.3 of this document entitled "BOND RX Protocol Setup Checklist: Imaging Round
 

13. Select "CST SignalStar" as preferred Detection System.
 

14. Select "Create Protocol."
 

15. Click Save and click Yes to acknowledge the caution message.

5 SignalStar Imaging Round 1: Solution Preparation

Warning

Each reagent MUST be used in the appropriate BOND RX container or insert from the BOND Titration Kit (#OPT9049) for the assay to run properly. See the table below for the number of containers or inserts necessary to perform the SignalStar assay on the BOND RX autostainer.
Warning

DO NOT OVERFILL OPEN CONTAINERS. BOND RX autostainer will consider containers to be "Empty" if they are overfilled.

 

Reagent
BOND Container		 Number of Containers Required
5 Slides 10 Slides
SignalStar Imaging Round 1 Solution BOND 6 mL Titration Inserts 1 1
SignalStar Amplification Solution 1 BOND 6 mL Titration Inserts 1 2
SignalStar Amplification Solution 2 BOND 6 mL Titration Inserts 1 2
SignalStar Ligation Solution BOND 6 mL Titration Inserts 1 1
10% Neutral Buffered Formalin BOND 7 mL Open Container 1 1
0.1X TBST Solution BOND 30 mL Open Container 2 3
1X TBST Solution BOND 30 mL Open Container 1 1

5.1 Imaging Round 1: SignalStar Solution

Caution

Once prepared, the SignalStar Imaging Round 1 Solution should contain ALL antibodies (up to 8) 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 10.1 and 9.1 of this document for guidance and assistance.
Warning

DO NOT COMBINE COMPLEMENTARY OLIGOS OF THE SAME FLUORESCENT CHANNEL.
Caution

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly into BOND 6 mL Titration Insert to ensure accuracy and avoid generating any bubbles. Store all SignalStar kit components on ice when preparing solutions. SignalStar solutions should be used promptly once all reagents have been combined for the run.

 

Kit Component		 Volume Required
5 Slides (µL) 10 Slides (µL)
SignalStarAntibody Diluent A 770 1,295
SignalStarAntibody Diluent B 330 555
SignalStarOligo-Conjugated Antibody 1 11 18.5
SignalStarOligo-Conjugated Antibody 2 11 18.5
SignalStarOligo-Conjugated Antibody 3 11 18.5
SignalStarOligo-Conjugated Antibody 4 (if required) 11 18.5
SignalStarOligo-Conjugated Antibody 5 (if required) 11 18.5
SignalStarOligo-Conjugated Antibody 6 (if required) 11 18.5
SignalStarOligo-Conjugated Antibody 7 (if required) 11 18.5
SignalStarOligo-Conjugated Antibody 8 (if required) 11 18.5
Imaging Round 1: SignalStar Complementary Oligo 1 11 18.5
Imaging Round 1: SignalStar Complementary Oligo 2 11 18.5
Imaging Round 1: SignalStar Complementary Oligo 3 11 18.5
Imaging Round 1: SignalStar Complementary Oligo 4 (if required) 11 18.5
Total Volume 1,232 2,072

5.2 Imaging Round 1: SignalStar Amplification Solution 1

Caution

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly into BOND 6 mL Titration Insert to ensure accuracy and avoid generating any bubbles. Store all SignalStar kit components on ice when preparing solutions. SignalStar solutions should be used promptly once all reagents have been combined for the run.

Caution

For a 10 slide run, make up the total volume in a conical tube, rotate for 20 min, then divide evenly among 2 titration inserts.
 

Kit Component		 Volume Required
5 Slides (µL) 10 Slides (µL)
SignalStar Amplification Buffer A 2,425 4,675
SignalStar Amplification Buffer B 2,425 4,675
SignalStar Amplification Oligo A-488 48.5 93.5
SignalStar Amplification Oligo A-594 48.5 93.5
SignalStar Amplification Oligo A-647 48.5 93.5
SignalStar Amplification Oligo A-750 48.5 93.5
Total Volume 5,044 9,724

5.3 Imaging Round 1: SignalStar Amplification Solution 2

Caution

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly into BOND 6 mL Titration Insert to ensure accuracy and avoid generating any bubbles. Store all SignalStar kit components on ice when preparing solutions. SignalStar solutions should be used promptly once all reagents have been combined for the run.

Caution

For a 10 slide run, make up the total volume in a conical tube, rotate for 20 min, then divide evenly among 2 titration inserts.
 

Kit Component		 Volume Required
5 Slides (µL) 10 Slides (µL)
SignalStar Amplification Buffer A 2,425 4,675
SignalStar Amplification Buffer B 2,425 4,675
SignalStar Amplification Oligo B-488 48.5 93.5
SignalStar Amplification Oligo B-594 48.5 93.5
SignalStar Amplification Oligo B-647 48.5 93.5
SignalStar Amplification Oligo B-750 48.5 93.5
Total Volume 5,044 9,724

5.4 Imaging Round 1: SignalStar Ligation Solution

Caution

Combine the following reagents in one BOND Titration Insert. Cover with parafilm and vortex for 10 sec. Store all SignalStar kit components on ice when preparing solutions. SignalStar solutions should be used promptly once all reagents have been combined for the run.

 

Kit Components/Reagents		 Volume Required
5 Slides (µL) 10 Slides (µL)
SignalStar Ligation Buffer 550 925
Nuclease-free Water #12931 517 869.5
T4 DNA Ligase (5 U/µL) 22 37
ATP (100 mM) 11 18.5
Total Volume 1,100 1,850

5.5 Additional Required Solutions Not Included

 
  • 0.1X Tris Buffered Saline with Tween 20 (TBST): To prepare 500 mL of 1X TBST, add 5 mL Tris Buffered Saline with Tween 20 (TBST-10X) #9997 to 495 mL dH2O. Fill 30 mL BOND Open Containers (2 containers for 5 slides, and 3 containers for 10 slides). Avoid generating bubbles during preparation.
  • 1X Tris Buffered Saline with Tween 20 (TBST): To prepare 500 mL of 1X TBST, add 50 mL Tris Buffered Saline with Tween 20 (TBST-10X) #9997 to 450 mL dH2O. Fill 30 mL BOND Open Container (1 container for 5 slides or 10 slides). Avoid generating bubbles during preparation.
  • 10% Neutral Buffered Formalin: Fill a 7 mL BOND Open Container, working in a laboratory fume hood.

6 SignalStar Imaging Round 1: BOND RX Protocol for Use

6.1 Slide Baking

Note

Slide baking can be performed the day before you begin your experiment. This step allows for the paraffin wax to melt.
  1. Incubate slides for 30 min at 60°C.

6.2 Running the BOND RX Protocol

  2. In the BOND RX software, create study and add slides.
  3. When "adding slides," use the below selections for Tissue Preparation on BOND:
  1. Slide preparation: Dewax
  2. Dispense Volume: 150 µL
  3. HIER: ER2 40 min
  4. Select "SignalStar Imaging Round 1" as the protocol and "HIER ER2 40 min" for each slide.
  5. Print labels, apply to slides, and add slides to the BOND slide tray.
  6. Place BOND covertiles on slides, ensuring that they are properly seated in the tray.
Caution

It is good practice to confirm that each step in the SignalStar Imaging Round 1 protocol is correct. Please use the checklist in section 9.2 to assist in creating your protocol.
Caution

Ensure that selected 0.1X TBST washes listed below are set to "Open." Fluorescent signals may be variable or diminished if washes are not "Open." Confirm that there are a total of 6 SignalStar Amplification Solution 1 steps and 6 SignalStar Amplification Solution 2 steps.
Caution

Confirm the bulk Bond Wash Solution container is full, and the waste containers are empty.
  7. Start BOND RX run.
Warning

Immediately start the BOND RX staining run, do not use a delayed start. Slides can sit overnight on the BOND RX autostainer once staining is complete.
  8. Remove slides from the BOND RX autostainer and place into 0.1X TBST.
  9. Run aspirating probe cleaning run on the BOND RX autostainer. Clean covertiles as per routine procedures.
  10. Prepare DAPI #4083 solution according to datasheet instructions.
  11. Counterstain with DAPI solution.
  12. Immerse slides in 0.1X TBST for 30 sec.
  13. Mount slides with ProLong Gold Antifade Reagent #9071.
  14. Image slides within 8 hr.
Caution

Do not let slides dry out at any point once they are deparaffinized or have had coverslips removed.

7 SignalStar Imaging Round 2: Solution Preparation

Warning

Each reagent MUST be used in the appropriate BOND RX container or insert from the BOND Titration Kit (#OPT9049) for the assay to run properly. See the table below for the number of containers or inserts necessary to perform the SignalStar assay on the BOND RX autostainer.
Warning

DO NOT OVERFILL OPEN CONTAINERS. BOND RX autostainer will consider containers to be "Empty" if they are overfilled.

 

Reagent
BOND Container		 Number of Containers Required
5 Slides 10 Slides
SignalStar Imaging Round 2 Solution BOND 6 mL Titration Inserts 1 1
SignalStar Amplification Solution 1 BOND 6 mL Titration Inserts 1 2
SignalStar Amplification Solution 2 BOND 6 mL Titration Inserts 1 2
SignalStar Ligation Solution BOND 6 mL Titration Inserts 1 1
10% Neutral Buffered Formalin BOND 7 mL Open Container 1 1
0.1X TBST Solution BOND 30 mL Open Container 2 3
1X TBST Solution BOND 30 mL Open Container 1 1
SignalStar Fluorescent Removal Solution BOND 6 mL Titration Insterts 1 1

7.1 Imaging Round 2: SignalStar Solution

Caution

Once prepared, the SignalStar Imaging Round 2 Solution should contain the complementary oligos for Imaging Round 2. Utilize the Example SignalStar Panel Design and SignalStar Panel Design Worksheet in section 10.1 and 9.1 of this document for guidance and assistance.
Warning

DO NOT COMBINE COMPLEMENTARY OLIGOS OF THE SAME FLUORESCENT CHANNEL.
Caution

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly into BOND 6 mL Titration Insert to ensure accuracy and avoid generating any bubbles. Store all SignalStar kit components on ice when preparing solutions. SignalStar solutions should be used promptly once all reagents have been combined for the run.
 

Kit Components		 Volume Required
5 Slides (µL) 10 Slides (µL)
SignalStar Antibody Diluent A 770 1,295
SignalStar Antibody Diluent B 330 555
Imaging Round 2: SignalStar Complementary Oligo 5 11 18.5
Imaging Round 2: SignalStar Complementary Oligo 6 (if required) 11 18.5
Imaging Round 2: SignalStar Complementary Oligo 7 (if required) 11 18.5
Imaging Round 2: SignalStar Complementary Oligo 8 (if required) 11 18.5
Total Volume 1,144 1,924

7.2 Imaging Round 2: SignalStar Amplification Solution 1

Caution

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly into BOND 6 mL Titration Insert to ensure accuracy and avoid generating any bubbles. Store all SignalStar kit components on ice when preparing solutions. SignalStar solutions should be used promptly once all reagents have been combined for the run.

Caution

For a 10 slide run, make up the total volume in a conical tube, rotate for 20 min, then divide evenly among 2 titration inserts.

 

Kit Components		 Volume Required
5 Slides (µL) 10 Slides (µL)
SignalStar Amplification Buffer A 2,425 4,675
SignalStar Amplification Buffer B 2,425 4,675
SignalStar Amplification Oligo A-488 48.5 93.5
SignalStar Amplification Oligo A-594 48.5 93.5
SignalStar Amplification Oligo A-647 48.5 93.5
SignalStar Amplification Oligo A-750 48.5 93.5
Total Volume 5,044 9,724

7.3 Imaging Round 2: SignalStar Amplification Solution 2

Caution

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly into BOND 6 mL Titration Insert to ensure accuracy and avoid generating any bubbles. Store all SignalStar kit components on ice when preparing solutions. SignalStar solutions should be used promptly once all reagents have been combined for the run.

Caution

For a 10 slide run, make up the total volume in a conical tube, rotate for 20 min, then divide evenly among 2 titration inserts.

 

Kit Components		 Volume Required
5 Slides (µL) 10 Slides (µL)
SignalStar Amplification Buffer A 2,425 4,675
SignalStar Amplification Buffer B 2,425 4,675
SignalStar Amplification Oligo B-488 48.5 93.5
SignalStar Amplification Oligo B-594 48.5 93.5
SignalStar Amplification Oligo B-647 48.5 93.5
SignalStar Amplification Oligo B-750 48.5 93.5
Total Volume 5,044 9,724

7.4 Imaging Round 2: SignalStar Ligation Solution

Caution

Combine the following reagents in 1 BOND Titration Insert. Cover with parafilm and vortex for 10 sec. Store all SignalStar kit components on ice when preparing solutions. SignalStar solutions should be used promptly once all reagents have been combined for the run.

 

Kit Components/Reagents		 Volume Required
5 Slides (µL) 10 Slides (µL)
SignalStar Ligation Buffer 550 925
Nuclease-free Water #12931 517 869.5
T4 DNA Ligase (5 U/µL) 22 37
ATP (100 mM) 11 18.5
Total Volume 1,100 1,850

7.5 Imaging Round 2: SignalStar Fluorescent Removal Solution

Caution

Combine the following reagents in 1 BOND Titration Insert. Cover with parafilm and vortex for 10 sec. Store all SignalStar kit components on ice when preparing solutions. SignalStar solutions should be used promptly once all reagents have been combined for the run.
 

Kit Components/Reagents		 Volume Required
5 Slides (µL) 10 Slides (µL)
10X dsDNase Buffer 185 335
dsDNase 18.5 33.5
Nuclease-free Water #12931 1,646.5 2,981.5
Total Volume 1,850 3,350

7.6 Additional Required Solutions Not Included

 
  • 0.1X Tris Buffered Saline with Tween 20 (TBST): To prepare 500 mL of 1X TBST, add 5 mL Tris Buffered Saline with Tween 20 (TBST-10X) #9997 to 495 mL dH2O. Fill 30 mL BOND Open Containers (2 containers for 5 slides, and 3 containers for 10 slides). Avoid generating bubbles during preparation.
  • 1X Tris Buffered Saline with Tween 20 (TBST): To prepare 500 mL of 1X TBST, add 50 mL Tris Buffered Saline with Tween 20 (TBST-10X) #9997 to 450 mL dH2O. Fill 30 mL BOND Open Container (1 container for 5 slides or 10 slides). Avoid generating bubbles during preparation.
  • 10% Neutral Buffered Formalin: Fill a 7 mL BOND Open Container, working in a laboratory fume hood.

8 SignalStar Imaging Round 2: BOND RX Protocol for Use

  1. After image acquisition of SignalStar Imaging Round 1, soak slides in dH2O for >30 min to gently remove coverslip.
Caution

Do not let slides dry out at any point once they are deparaffinized or have had coverslips removed.
  2. In the BOND RX software, create study and add slides.
  3. When "adding slides," use the below selections for Tissue Preparation on BOND:
  1. Slide preparation: -- (no value/not required)
  2. Dispense Volume: 150 µL
  3. HIER: -- (no value/not required)
  4. Select "CST SignalStar Imaging Round 2," ensuring that Slide preparation is selected as "--" and HIER is selected as "--."
  5. Print labels, add labels to slides, and place slides onto the slide tray.
  6. Place 2-3 drops of dH2O onto each slide before adding BOND covertiles.
Warning

IMPORTANT: Dewax and HIER (antigen retrieval) are not required for Amplification Round 2. If Dewax and HIER are used, Amplification Round 2 results will be uninterpretable.

Caution

It is good practice to confirm that each step in the Imaging Round 2 protocol is correct. Please use the checklist in section 9.3 to assist in creating your protocol.
Caution

Ensure that selected 0.1X TBST washes listed below are set to "Open." Fluorescent signals may be variable or diminished if washes are not "Open." Confirm that there are a total of 6 SignalStar Amplification Solution 1 steps and 6 SignalStar Amplification Solution 2 steps.
Caution

Confirm the bulk Bond Wash Solution container is full, and the waste containers are empty.
  7. Start the BOND RX run.
Warning

Immediately start the BOND RX staining run, do not use a delayed start. Slides can sit overnight on the BOND RX autostainer once staining is complete.
  8. Remove slides from the BOND RX autostainer and place into 0.1X TBST.
  9. Run aspirating probe cleaning run on the BOND RX autostainer. Clean covertiles as per routine procedures.
  10. Prepare DAPI #4083 solution according to datasheet instructions.
  11. Counterstain with DAPI solution.
  12. Immerse slides in 0.1X TBST for 30 sec.
  13. Mount slides with ProLong Gold Antifade Reagent #9071.
  14. Image slides within 8 hr.
Caution

Do not let slides dry out at any point once they are deparaffinized or have had coverslips removed.

9 User Worksheets

9.1 SignalStar Panel Design Worksheet

Oligo-Conjugated Antibody Complementary Oligo Product Pair # Imaging Round 488 594 647 750
               
               
               
               
               
               
               
               

(See Appendix 10.1 for an example panel design.)

9.2 BOND RX Protocol Setup Checklist: Imaging Round 1

BOND Step ✓ Reagent Step Type Incubation Time (min) Temperature (C) Dispense Type
1 ☐ *Deionized Water Wash 0:00 Ambient 150 µL
2 ☐ *Deionized Water Wash 0:00 Ambient Open
3 ☐ *Deionized Water Wash 0:00 Ambient 150 µL
4 ☐ 0.1X TBST Solution Wash 0:00 Ambient 150 µL
5 ☐ MARKER (SignalStar Imaging Round 1 Solution) Primary 40:00 Ambient 150 µL
6 ☐ 1X TBST Solution Reagent 5:00 Ambient Open
7 ☐ 0.1X TBST Solution Reagent 5:00 Ambient 150 µL
8 ☐ 10% Neutral Buffered Formalin Reagent 5:00 Ambient 150 µL
9 ☐ *Deionized Water Wash 0:00 Ambient Open
10 ☐ *Deionized Water Wash 0:00 Ambient Open
11 ☐ *Deionized Water Wash 0:00 Ambient 150 µL
12 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
13 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
14 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
15 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
16 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
17 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
18 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
19 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
20 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
21 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
22 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
23 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
24 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
25 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
26 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
27 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
28 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
29 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
30 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
31 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
32 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
33 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
34 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
35 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
36 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
37 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
38 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
39 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
40 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
41 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
42 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
43 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
44 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
45 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
46 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
47 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
48 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
49 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
50 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
51 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
52 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
53 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
54 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
55 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
56 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
57 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
58 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
59 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
60 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
61 ☐ SignalStar Ligation Solution Reagent 20:00 Ambient 150 µL
62 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
63 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
64 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
65 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
66 ☐ BOND Wash Solution Wash 0:00 Ambient 150 µL

9.3 BOND RX Protocol Setup Checklist: Imaging Round 2

BOND Step ✓ Reagent Step Type Incubation Time (min) Temperature (C) Dispense Type
1 ☐ 0.1X TBST Solution Wash 0:00 Ambient Open
2 ☐ 0.1X TBST Solution Wash 0:00 Ambient Open
3 ☐ SignalStar Stripping Buffer Reagent 60:00 37° 150 µL
4 ☐ SignalStar Stripping Buffer Reagent 60:00 37° 150 µL
5 ☐ *Deionized Water Wash 0:00 Ambient Open
6 ☐ *Deionized Water Wash 0:00 Ambient Open
7 ☐ *Deionized Water Wash 0:00 Ambient Open
8 ☐ MARKER (SignalStar Imaging Round 2 Solution) Primary 40:00 Ambient 150 µL
9 ☐ 1X TBST Solution Reagent 5:00 Ambient Open
10 ☐ 0.1X TBST Solution Reagent 5:00 Ambient Open
11 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
12 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
13 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
14 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
15 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
16 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
17 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
18 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
19 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
20 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
21 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
22 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
23 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
24 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
25 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
26 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
27 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
28 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
29 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
30 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
31 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
32 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
33 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
34 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
35 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
36 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
37 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
38 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
39 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
40 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
41 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
42 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
43 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
44 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
45 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
46 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
47 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
48 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
49 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
50 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
51 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
52 ☐ SignalStar Amplification Solution 1 Reagent 8:00 Ambient 150 µL
53 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
54 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
55 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
56 ☐ SignalStar Amplification Solution 2 Reagent 16:00 Ambient 150 µL
57 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
58 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
59 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
60 ☐ SignalStar Ligation Solution Reagent 20:00 Ambient 150 µL
61 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
62 ☐ 0.1X TBST Solution Reagent 0:00 Ambient Open
63   BOND Wash Solution Wash 0:00 Ambient 150 µL

10 Appendix

10.1 Example SignalStar Panel Design

Oligo-Conjugated Antibody Complementary Oligo Product Pair # Imaging Round 488 594 647 750
PD-1 (Intracellular Domain) (D4W2J)
XP® Rabbit mAb
(SignalStar™ Conjugate 0008)		 Complementary Oligo
(CO-0008-488)		 17942 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       ●

10.2 Troubleshooting and Frequently Asked Questions

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.

Does this assay work on frozen tissue?

SignalStar Multiplex IHC Kits & Reagents haven't yet been validated for use in frozen tissues. We're in the process of validating our antibodies and protocols for use in fresh or frozen tissue.

Do you have anti-mouse antibodies available?

SignalStar Multiplex IHC Kits & Reagents haven't yet been validated for use in mouse tissues. We're in the process of validating mouse-reactive antibodies.

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.

Can I combine antibodies used in this assay with direct conjugates?

SignalStar Multiplex IHC Kits & Reagents haven't been validated for use in combination with direct conjugates. It's likely possible to incorporate direct conjugates into your protocol. However, because the SignalStar reagents benefit from fluorescent signal amplification, there may be spectral bleed-through that results from using the assay with conjugates that are not amplified.

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.

For Research Use Only. Not for Use in Diagnostic Procedures.

Cell Signaling Technology, XP, and SignalStar are trademarks of Cell Signaling Technology, Inc.
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posted July 2023

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