Granzyme B (D6E9W) & CO-0009-647 SignalStar^™ Oligo-Antibody Pair #37369

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.

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.

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.

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

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.

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.

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.

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

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.

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

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.

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.

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.

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.

1. MARKER (= SignalStar Staining Solution)

2. SignalStar Amplification Solution 1

3. SignalStar Amplification Solution 2

4. SignalStar Ligation Solution

5. SignalStar Fluorescent Removal Solution

6. 10% Neutral Buffered Formalin

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

7. 0.1X TBST

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

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.

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

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."

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."

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

14. Select "Create Protocol."

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

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.

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

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.

DO NOT COMBINE COMPLEMENTARY OLIGOS OF THE SAME FLUORESCENT CHANNEL.

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.

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.

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.

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.

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.

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.

• 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.

Slide baking can be performed the day before you begin your experiment. This step allows for the paraffin wax to melt.

1. Slide preparation: Dewax
2. Dispense Volume: 150 µL
3. HIER: ER2 40 min

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.

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.

Confirm the bulk Bond Wash Solution container is full, and the waste containers are empty.

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.

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

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.

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

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.

DO NOT COMBINE COMPLEMENTARY OLIGOS OF THE SAME FLUORESCENT CHANNEL.

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.

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.

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.

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.

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.

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.

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.

• 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.

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

1. Slide preparation: -- (no value/not required)
2. Dispense Volume: 150 µL
3. HIER: -- (no value/not required)

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.

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.

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.

Confirm the bulk Bond Wash Solution container is full, and the waste containers are empty.

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.

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

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.

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.

Please confirm whether your SignalStar panel design requires 2 rounds of imaging. Utilize the SignalStar Panel Design and SignalStar Panel Design Worksheet in section 9.1 and 8.1 of this document for guidance and assistance.

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.

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.

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

Fluorescent signal may be variable or diminished if solutions are not sufficiently mixed. Combine SignalStar kit components using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when not in use. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

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

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.

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.

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.

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.

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 9.1 and 8.1 of this document for guidance and assistance.

DO NOT COMBINE COMPLEMENTARY OLIGOS OF THE SAME FLUORESCENT CHANNEL.

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

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.

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.

2. Incubate sections in 3 washes of xylene for 5 min each.

3. Incubate sections in 2 washes of 100% ethanol for 10 min each.

4. Incubate sections in 2 washes of 95% ethanol for 10 min each.

5. Wash sections 2 times in dH₂O for 5 min each.

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 and operating instructions should be utilized for other pressure cookers.

7. Place 500 mL dH₂O into the pressure cooker.

8. Place the slide holder into the pressure cooker, touching the heat shield. Partially cover with a slide container lid.

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.

9. Seal the chamber and proceed with retrieval. Settings for the Biocare Medical Decloaking Chamber #DC2012 are as follows:

1. SP1 125°C for 30 sec
2. SP2 90°C for 10 sec

10. Carefully vent the device, then remove the lid.

11. Remove slides from the decloaking chamber and allow to cool on the bench top for 10 min.

13. Incubate slides in 150 µL of SignalStar Imaging Round 1 Solution for 40 min at room temperature.

Slides can alternatively be incubated in SignalStar Imaging Round 1 Solution overnight at 4°C in order to break up the protocol into multiple days.

14. Thoroughly flick off liquid from slides and immerse in 1X TBST for 30 sec.

15. Incubate slides in 10% Neutral Buffered Formalin for 5 min at room temperature.

16. Thoroughly flick off liquid from slides and immerse in dH₂O for 30 sec.

1. Incubate slides in 150 µL of Amplification Solution 1 for 8 min.
2. Thoroughly flick off liquid from slides and immerse in dH₂O for 30 sec.
3. Incubate slides in 150 µL of Amplification Solution 2 for 8 min.
4. Thoroughly flick off liquid from slides and immerse in dH₂O for 30 sec.

18. Incubate slides in 150 µL of SignalStar Ligation Buffer for 20 min at room temperature.

19. Thoroughly flick off liquid from slides and immerse in dH₂O for 30 sec.

20. Prepare DAPI #4083 solution according to datasheet instructions.

21. Immerse slides in 1X TBST for 30 sec.

22. Counterstain with DAPI solution.

23. Immerse slides in 1X TBST for 30 sec.

24. Mount slides with ProLong Gold Antifade Reagent #9071.

25. Image slides as soon as possible. Signal should remain robust for up to 8 hr.

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.

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

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.

NO ANTIBODY-OLIGO CONJUGATES ARE ADDED TO THE SIGNALSTAR IMAGING ROUND 2 SOLUTION.

DO NOT COMBINE COMPLEMENTARY OLIGOS OF THE SAME FLUORESCENT CHANNEL.

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

Prepare solutions using low retention pipette tips and rotate end-over-end for 20 min at room temperature. Pipette slowly to ensure accuracy. Store all SignalStar kit components on ice when preparing solutions. Once combined, SignalStar solutions should be kept at room temperature and used promptly.

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.

Once imaging is complete, fluorescent signal can be removed from slides so that another round of imaging can be performed. Perform the next set of steps as soon as possible following the first round of imaging.

1. After image acquisition, soak slides in dH₂O for ≥30 min to gently remove coverslips without damaging tissue.

2. Incubate slides in 150 µL of SignalStar Fluorescent Removal Solution for 2 hr at 37°C.

3. Immerse slides in dH₂O for 30 sec.

4. Optional:

1. Mount slides in ProLong Gold Antifade Reagent #9071 with DAPI #4083 and image slides to confirm that no fluorescent signal remains.
2. Soak slides in dH₂O for ≥ 30 min to gently remove coverslips without damaging tissue.
3. Immerse slides in dH₂O for 30 sec.

5. Incubate slides in 150 µL of SignalStar Imaging Round 2 Solution for 40 min at room temperature.

6. Thoroughly flick off liquid from slides and immerse in dH₂O for 30 sec.

1. Incubate slides in 150 µL of Amplification Solution 1 for 8 min.
2. Thoroughly flick off liquid from slides and immerse in dH₂O for 30 sec.
3. Incubate slides in 150 µL of Amplification Solution 2 for 8 min.
4. Thoroughly flick off liquid from slides and immerse in dH₂O for 30 sec.

8. Incubate slides in 150 µL of SignalStar Ligation Buffer for 20 min at room temperature.

9. Thoroughly flick off liquid from slides and immerse in dH₂O for 30 sec.

10. Prepare DAPI #4083 solution according to datasheet instructions.

11. Immerse slides in 1X TBST for 30 sec.

12. Counterstain with DAPI solution.

13. Immerse slides in 1X TBST for 30 sec.

14. Thoroughly flick off liquid from slides and immerse in dH₂O for 30 sec.

15. Mount slides with ProLong Gold Antifade Reagent #9071.

16. Image slides as soon as possible. Signal should remain robust for up to 8 hr.