Cell Signaling Technology^® SignalStar^™ Multiplex Immunohistochemistry Assay Manual

Product Information:

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.

1 Introduction: The SignalStar™ Staining Methodology
2 Solutions and Reagents
- 2.1 Included Kit Components
- 2.2 Required Reagents Not Included
3 Important Considerations Before You Begin
4 SignalStar Imaging Round 1: Solution Preparation
5 SignalStar Imaging Round 1: Protocol for Use
6 SignalStar Imaging Round 2: Solution Preparation
7 SignalStar Imaging Round 2: Protocol for Use
8 User Worksheets
9 Appendix
- 9.1 Example SignalStar Panel Design
- 9.2 Troubleshooting and Frequently Asked Questions

1 Introduction: The SignalStar™ Staining Methodology

SignalStar™ Multiplex Immunohistochemistry (mIHC) is a technology that employs antibodies, oligonucleotides (oligos), and fluorophores to interrogate 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

SignalStain^® EDTA Unmasking Solution (10X) #14747
Xylene (for deparaffinization)
Ethanol, anhydrous denatured, histological grade (100% and 95%)
Decloaking Chamber (Biocare Medical, #DC2012)
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
Slide Processing Containers
Hydrophobic Barrier Pen
Glass Coverslips
Charged Slides
Control Tissues

3 Important Considerations Before You Begin

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.

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

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.

4 SignalStar Imaging Round 1: Solution Preparation

4.1 Imaging Round 1: SignalStar Solution

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.

Kit Component	Volume Required
Kit Component	5 Slides (µL)	10 Slides (µL)
SignalStarAntibody Diluent A	525	1,050
SignalStarAntibody Diluent B	225	450
SignalStarOligo-Conjugated Antibody 1	7.5	15
SignalStarOligo-Conjugated Antibody 2	7.5	15
SignalStarOligo-Conjugated Antibody 3	7.5	15
SignalStarOligo-Conjugated Antibody 4 (if required)	7.5	15
SignalStarOligo-Conjugated Antibody 5 (if required)	7.5	15
SignalStarOligo-Conjugated Antibody 6 (if required)	7.5	15
SignalStarOligo-Conjugated Antibody 7 (if required)	7.5	15
SignalStarOligo-Conjugated Antibody 8 (if required)	7.5	15
Imaging Round 1: SignalStar Complementary Oligo 1	7.5	15
Imaging Round 1: SignalStar Complementary Oligo 2	7.5	15
Imaging Round 1: SignalStar Complementary Oligo 3	7.5	15
Imaging Round 1: SignalStar Complementary Oligo 4 (if required)	7.5	15
Total Volume	840	1,680

4.2 Imaging Round 1: SignalStar Amplification Solution 1

Kit Component	Volume Required
Kit Component	5 Slides (µL)	10 Slides (µL)
SignalStar Amplification Buffer A	3,000	6,000
SignalStar Amplification Buffer B	3,000	6,000
SignalStar Amplification Oligo A-488	60	120
SignalStar Amplification Oligo A-594	60	120
SignalStar Amplification Oligo A-647	60	120
SignalStar Amplification Oligo A-750	60	120
Total Volume	6,240	12,480

4.3 Imaging Round 1: SignalStar Amplification Solution 2

Kit Component	Volume Required
Kit Component	5 Slides (µL)	10 Slides (µL)
SignalStar Amplification Buffer A	3,000	6,000
SignalStar Amplification Buffer B	3,000	6,000
SignalStar Amplification Oligo B-488	60	120
SignalStar Amplification Oligo B-594	60	120
SignalStar Amplification Oligo B-647	60	120
SignalStar Amplification Oligo B-750	60	120
Total Volume	6,240	12,480

4.4 Imaging Round 1: SignalStar Ligation Solution

Kit Components/Reagents	Volume Required
Kit Components/Reagents	5 Slides (µL)	10 Slides (µL)
SignalStar Ligation Buffer	375	750
Nuclease-free Water #12931	352.5	705
T4 DNA Ligase (5 U/µL)	15	30
ATP (100 mM)	7.5	15
Total Volume	750	1,500

4.5 Additional Required Solutions Not Included

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.
10% Neutral Buffered Formalin
1X Tris Buffered Saline with Tween 20 (TBST): To prepare 1 L 1X TBST, add 10 mL Tris Buffered Saline with Tween 20 (TBST-10X) #9997 to 900 mL of dH₂O, mix.

5 SignalStar Imaging Round 1: Protocol for Use

5.1 Slide Baking

	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.

5.2 Deparaffinization/Hydration

	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.

5.3 Antigen Unmasking

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.

6.	Place slides in 250 mL room temperature 1X EDTA Unmasking Solution in a 24-slide holder. Add blank slides to completely fill the slide holder.

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:

SP1 125°C for 30 sec
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.

12.	Place the container of slides under a dH₂O faucet and add water directly to the slide container until all EDTA is replaced by dH₂O.

5.4 Imaging Round 1: Staining

	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.

5.5 Imaging Round 1: Amplification

17.	Perform 8 rounds of amplification with Amplification Solutions 1 and 2. Use the SignalStar Imaging Round 1 Checklist in section 8.2 to track your progress. Each amplification round consists of the following steps:

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

5.6 Imaging Round 1: Ligation

	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.

5.7 Imaging Round 1: Image

	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.

6 SignalStar Imaging Round 2: Solution Preparation

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.

6.1 Imaging Round 2: SignalStar Fluorescent Removal Solution

Kit Components/Reagents	Volume Required
Kit Components/Reagents	5 Slides (µL)	10 Slides (µL)
10X dsDNase Buffer	75	150
dsDNase	7.5	15
Nuclease-free Water #12931	667.5	1,335
Total Volume	750	1,500

6.2 Imaging Round 2: SignalStar Solution

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.

Kit Components	Volume Required
Kit Components	5 Slides (µL)	10 Slides (µL)
SignalStar Antibody Diluent A	525	1,050
SignalStar Antibody Diluent B	225	450
Imaging Round 2: SignalStar Complementary Oligo 5	7.5	15
Imaging Round 2: SignalStar Complementary Oligo 6 (if required)	7.5	15
Imaging Round 2: SignalStar Complementary Oligo 7 (if required)	7.5	15
Imaging Round 2: SignalStar Complementary Oligo 8 (if required)	7.5	15
Total Volume	780	1,560

6.3 Imaging Round 2: SignalStar Amplification Solution 1

Kit Components	Volume Required
Kit Components	5 Slides (µL)	10 Slides (µL)
SignalStar Amplification Buffer A	3,000	6,000
SignalStar Amplification Buffer B	3,000	6,000
SignalStar Amplification Oligo A-488	60	120
SignalStar Amplification Oligo A-594	60	120
SignalStar Amplification Oligo A-647	60	120
SignalStar Amplification Oligo A-750	60	120
Total Volume	6,240	12,480

6.4 Imaging Round 2: SignalStar Amplification Solution 2

Kit Components	Volume Required
Kit Components	5 Slides (µL)	10 Slides (µL)
SignalStar Amplification Buffer A	3,000	6,000
SignalStar Amplification Buffer B	3,000	6,000
SignalStar Amplification Oligo B-488	60	120
SignalStar Amplification Oligo B-594	60	120
SignalStar Amplification Oligo B-647	60	120
SignalStar Amplification Oligo B-750	60	120
Total Volume	6,240	12,480

6.5 Imaging Round 2: SignalStar Ligation Solution

Kit Components/Reagents	Volume Required
Kit Components/Reagents	5 Slides (µL)	10 Slides (µL)
SignalStar Ligation Buffer	375	750
Nuclease-free Water #12931	352.5	705
T4 DNA Ligase (5 U/µL)	15	30
ATP (100 mM)	7.5	15
Total Volume	750	1,500

7 SignalStar Imaging Round 2: Protocol for Use

	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.

7.1 Removal of Fluorescent Signal

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

7.2 Imaging Round 2: Staining

	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.

7.3 Imaging Round 2: Amplification

7.	Perform 8 rounds of amplification with Amplification Solutions 1 and 2. Use the SignalStar Imaging Round 2 Checklist in section 8.3 to track your progress. Each amplification round consists of the following steps:

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

7.4 Imaging Round 2: Ligation

	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.

7.5 Imaging Round 2: Image

	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.

8 User Worksheets

8.1 SignalStar Panel Design Worksheet

Oligo-Conjugated Antibody	Complementary Oligo	Imaging Round	488	594	647	750

(See Appendix 9.1 for an example panel design.)

8.2 SignalStar Imaging Round 1 Checklist

Amplification Round	Step #	✓	Step
Amplification Round 1	1	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	2	☐	Immerse slides in dH₂O for 30 sec.
	3	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	4	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 2	5	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	6	☐	Immerse slides in dH₂O for 30 sec.
	7	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	8	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 3	9	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	10	☐	Immerse slides in dH₂O for 30 sec.
	11	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	12	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 4	13	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	14	☐	Immerse slides in dH₂O for 30 sec.
	15	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	16	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 5	17	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	18	☐	Immerse slides in dH₂O for 30 sec.
	19	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	20	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 6	21	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	22	☐	Immerse slides in dH₂O for 30 sec.
	23	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	24	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 7	25	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	26	☐	Immerse slides in dH₂O for 30 sec.
	27	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	28	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 8	29	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	30	☐	Immerse slides in dH₂O for 30 sec.
	31	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	32	☐	Immerse slides in dH₂O for 30 sec.

8.3 SignalStar Imaging Round 2 Checklist

Amplification Round	Step #	✓	Step
Amplification Round 1	1	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	2	☐	Immerse slides in dH₂O for 30 sec.
	3	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	4	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 2	5	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	6	☐	Immerse slides in dH₂O for 30 sec.
	7	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	8	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 3	9	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	10	☐	Immerse slides in dH₂O for 30 sec.
	11	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	12	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 4	13	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	14	☐	Immerse slides in dH₂O for 30 sec.
	15	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	16	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 5	17	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	18	☐	Immerse slides in dH₂O for 30 sec.
	19	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	20	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 6	21	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	22	☐	Immerse slides in dH₂O for 30 sec.
	23	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	24	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 7	25	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	26	☐	Immerse slides in dH₂O for 30 sec.
	27	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	28	☐	Immerse slides in dH₂O for 30 sec.
Amplification Round 8	29	☐	Incubate in SignalStar™ Amplification Solution 1 for 8 min.
	30	☐	Immerse slides in dH₂O for 30 sec.
	31	☐	Incubate in SignalStar™ Amplification Solution 2 for 8 min.
	32	☐	Immerse slides in dH₂O for 30 sec.

9 Appendix

9.1 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				●

9.2 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.

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posted July 2023

Cell Signaling Technology® SignalStar™ Multiplex Immunohistochemistry Assay Manual