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최근 각광받고 있는 Spatial Biology (공간생물학)은 조직 내에 특정 세포들이 어떻게 삼차원으로 분포되어 있는지를 관찰하여 기능 및 바이오마커의 발현 패턴과 상호작용을 연구하는 분야로 복잡한 세포와 조직, 질병의 이해와 치료를 위해 다양한 형태와 타겟을 대상으로 연구 기술이 개발되고 있습니다.
Cell Signaling Technology (CST)의 SignalStar Multiplex IHC는 Spatial Biology 연구를 위한 혁신적인 도구로, 고품질의 제품 및 검증된 항체 패널과 프로토콜을 제공합니다. SignalStar 기술은 높은 sensitivity와 specificity로 formalin-fixed, paraffin-embedded (FFPE) 조직 샘플에서 여러 바이오마커를 동시에 증폭합니다. 단 2일 만에 최대 8개의 target에 대한 정확하고 신뢰할 수 있는 IHC 데이터를 생성할 수 있습니다.
이제 CST의 신기술, SignalStar Multiplex IHC와 함께 빠르고 검증된 spatial biology 연구를 할 수 있습니다. 지금 여러분의 패널을 만들어보세요!
What is SignalStar Multiplex IHC?
SignalStar Multiplex IHC는 antibodies, oligonucleotides, 및 fluorophores을 사용하여 세포의 존재, 위치, 기능 및 바이오마커 동시 발현 패턴을 연구하는 기술입니다. SignalStar 분석을 사용하면 공간적 맥락과 조직 구조를 유지하면서 여러 표현형 및 기능적 targets을 검출할 수 있습니다. 이러한 특징은 세포가 조직 미세 환경에 영향을 미치고 질병 진행 및 치료 반응을 유도하기 위해 어떻게 조직되고 상호 작용하는지 이해하는 데 필수적입니다.
Why Choose Multiplex IHC?
Multiplex IHC (mIHC)는 광범위한 spatial biology 연구를 위한 다재다능하고 강력한 도구입니다. RNA 정보만으로는 충분하지 않기 때문에 target과 세포의 기본 상태를 이해해야 하거나, 하나의 조직 샘플에서 데이터 수집을 극대화하거나, 세포 기능을 이해하거나, 단백질을 검출해야 하는 경우 Multiplex IHC가 이상적인 해답이 될 수 있습니다.
- Understand biology and function in the native state
Multiplex IHC enables the simultaneous detection of multiple protein and modification biomarkers within a single tissue sample, maintaining the original spatial and cellular context of targets and providing insights into the underlying function of cells and tissues.
- Maximize data acquisition
Multiplex IHC lets you to maximize the amount of data acquired from an individual sample, reducing the need for additional tissue samples.
- Better post-translational insight
While RNA analysis can provide information about gene expression, it can’t capture post-translational protein modifications such as phosphorylation, glycosylation, and acetylation. Multiplex IHC allows for the detection of multiple protein modifications simultaneously, providing a more comprehensive understanding of the underlying biology.
How SignalStar Multiplex IHC Works
SignalStar 분석을 사용하면 FFPE 조직에서 최대 8개의 target을 동시에 검출할 수 있습니다. Deparaffinized 및 rehydrated FFPE 조직 절편은 antigen retrieval (A)를 거치며, 선택한 plex size의 모든 antibody (최대 3~8개의 고유한 oligo-conjugated antibodies)가 첫 incubation step (B)에 추가됩니다. Fluorescent dyes(채널: 488, 594, 647, 750nm)가 결합된 complementary oligonucleotides는 antibody에서 large oligo branched structures를 구축하여 첫 번째 round (imaging round 1)에서 최대 4개의 oligo-conjugated antibodies의 신호를 증폭합니다 (C-D). Plex size가 4개보다 큰 경우, imaging round 1의 oligonucleotides 와 fluorophores가 dsDNase에 의해 mild하게 제거되고(E), 두 번째 라운드 (imaging round 2)가 추가되어 최대 4개의 추가 oligo-conjugated antibodies(F)의 신호를 증폭합니다. 그런 다음 두 이미지는 소프트웨어를 사용하여 최대 8plex의 이미지(G)를 생성할 수 있습니다.
- Collection and fixation of the tissue is crucial as it directly affects sample integrity and experimental results. Tissue collection, preservation, and fixation procedures vary depending on the sample or the target of interest.
- Cross-linking fixatives, like formalin, are common for preparing samples for multiplex IHC as they preserve the structural integrity and architecture of the tissue.
- Fixatives used to preserve the structural integrity of the tissue sample may mask the epitope your antibody is designed to recognize.
- Several methods exist for revealing epitopes masked by fixation, including proteolytic-induced antigen retrieval or heat-induced epitope retrieval (HIER).
- Choose from a growing menu of antibodies validated to work in any combination with our universal manual and automated protocols. The fully validated protocols allow you to redesign your panels easily and work out of the box.
- All antibodies are added at once, eliminating antibody cycling and any possibility of epitope degradation or epitope masking during multiple rounds of imaging.
- A short post-stain fixation with 10% neutral buffered formalin guarantees antibodies remain on the slide during rounds of amplification and imaging.
- Amplify the fluorescent signal to enable detection of a wide, dynamic range of targets and outshine tissue autofluorescence.
- Complementary oligos specific to each antibody are added for the first 4 oligo-conjugated antibodies, followed by additional oligos with fluorophores to exponentially amplify the signal.
- Fluorophores used are spectrally distinct to lessen the need for spectral unmixing.
- Gentle removal of the oligos containing the fluorophores leaves the antibodies intact, enabling a second round of amplification and imaging.
- Pair any antibody with almost any fluorophore and run your experiment right out of the box!
SignalStar™ Multiplex IHC Panel Builder
SignalStar Multiplex IHC Panel Builder는 원활하고 간편한 멀티플렉스 IHC 패널 디자인을 제공합니다. 4가지 기본 단계를 통해 고객이 원하는 target species (2023년 9월 현재 human만 가능, mouse 업데이트 예정), SignalStar validated antibodies, fluorophores 및 이미징 시퀀스를 선택하여 고객 맞춤형 실험을 빠르고 쉽게 설계할 수 있습니다. 또는 고객이 원하는species과 antibodies를 선택하면 자동화된 panel builder가 나머지 과정을 안내할 수도 있습니다.
SignalStar™ Validated Antibodies (2023년 9월 현재, 지속적인 업데이트 예정)
| Antibodies | Clone | Antibodies | Clone | Antibodies | Clone |
| CD11b/ITGAM | D6X1N | CD68 | D4B9C | PD-L1 | E1L3N® |
| CD11c | D3V1E | CD86 | E2G8P | Pan-Keratin | C11 |
| CD16 | D1N9L | CD8α | D8A8Y | Phospho-Stat3 (Tyr705) | D3A7 |
| CD163 | D6U1J | Granzyme B | D6E9W | SIRPα/SHPS1 | D6I3M |
| CD20 | E7B7T | HLA-DRA | E9R2Q | TCF1/TCF7 | C63D9 |
| CD206/MRC1 | E2L9N | ICOS | D1K2T™ | TIM-3 | D5D5R™ |
| CD31 (PECAM-1) | 89C2 | Ki-67 | 8D5 | Tox/Tox2 | E6I3Q |
| CD3ε | D7A6E™ | LAG3 | D2G4O | Vimentin | D21H3 |
| CD44 | E7K2Y | NT5E/CD73 | D7F9A | α-Smooth Muscle Actin | D4K9N |
| CD45 (Intracellular Domain) | D9M8I | PD-1 (Intracellular Domain) | D4W2J |
SignalStar Multiplex IHC Benefits
Timelines for optimization and validation for other multiplex technologies can take weeks to months. SignalStar technology eliminates that time with optimized, ready-to-go panels. Select targets from a menu of IHC-validated antibodies that work out of the box in FFPE tissues.
Confidently analyze limited, precious FFPE tissue samples using highly-sensitive assays that give you the signal you need the first time. SignalStar assays enable you to amplify the fluorescent signal of up to 8 proteins in a single tissue.
Figure Legend: SignalStar multiplex immunohistochemical analysis of paraffin-embedded human gastric adenocarcinoma using Pan-Keratin (C11) & C0-0003-488 SignalStar Oligo-Antibody Pair #63566 (red), PD-L1 (E1L3N) & C0-0005-488 SignalStar Oligo-Antibody Pair #85646 (green), PD-1 (Intracellular Domain) (D4W2J) & C0-0008-594 SignalStar Oligo-Antibody Pair #35347 (yellow), Granzyme B (D6E9W) & C0-0009-594 SignalStar Oligo-Antibody Pair #15194 (orange), TIGIT (E5Y1W) & C0-0002-647 SignalStar Oligo-Antibody Pair #18288 (cyan), CD20 (E7B7T) & C0-0011-647 SignalStar Oligo-Antibody Pair #36775 (pink), CD3ε (D7A6E) Conjugate & C0-0001-750 SignalStar Oligo-Antibody Pair #51754 (magenta), CD8ɑ (D8A8Y) & C0-0004-750 SignalStar Oligo-Antibody Pair #62750 (white), and ProLong Gold Antifade Reagent with DAPI #8961 (blue). Representative individual and co-registered 8-plex images are included. All fluorophores were assigned a pseudocolor, as indicated. Staining was performed on the BOND RX autostainer by Leica Biosystems.
Many solutions lack the flexibility to evolve and adapt to changing research needs. SignalStar panels give you the flexibility to easily change targets and redesign panels as your needs change. Protocols are validated and optimized to work out of the box.
SignalStar technology stains consistently regardless of the panel you design in the SignalStar Multiplex IHC Panel Builder, and panels are ready to use out of the box. Protocols also demonstrate reproducibility across different antibody combinations, experiments, and imaging instruments.
Figure Legend:SignalStar multiplex immunohistochemical analysis of paraffin-embedded human medullary thyroid carcinoma within a tissue microarray. Panel 1: PD-L1 (E1L3N®) & C0-0005-647 SignalStar Oligo-Antibody Pair #52085 (yellow), PD-1 (Intracellular Domain) (D4W2J) & C0-0008-594 SignalStar Oligo-Antibody Pair #35347 (not shown), CD68 (D4B9C) & C0-0007-488 SignalStar Oligo-Antibody Pair #73071 (not shown), and CD3ε (D7A6E) & C0-0001-750 SignalStar Oligo-Antibody Pair #51754 (not shown). Panel 2: PD-L1 (E1L3N) & C0-0005-647 SignalStar Oligo-Antibody Pair #52085 (yellow), PD-1 (Intracellular Domain) (D4W2J) & C0-0008-594 SignalStar Oligo-Antibody Pair #35347 (not shown), CD68 (D4B9C) & C0-0007-488 SignalStar Oligo-Antibody Pair #73071 (not shown), and CD8 (D8A8Y) & C0-0004-750 SignalStar Oligo-Antibody Pair #62750 (not shown). Panel 3: PD-L1 (E1L3N) & C0-0005-647 SignalStar Oligo-Antibody Pair #52085 (yellow), PD-1 (Intracellular Domain) (D4W2J) & C0-0008-594 SignalStar Oligo-Antibody Pair #35347 (not shown), Ki-67 (8D5) & C0-0014-488 SignalStar Oligo-Antibody Pair #89034 (not shown), and CD8ɑ (D8A8Y) & C0-0004-750 SignalStar Oligo-Antibody Pair #62750 (not shown). All fluorophores were assigned a pseudocolor, as indicated. Staining was performed on the BOND RX autostainer. Percent positive PD-L1 cells were quantified in matched regions of interest from serial sections for three unique SignalStar 4-plex panels compared to single-plex SignalStar staining using PD-L1 (E1L3N) & C0-0005-647 SignalStar Oligo Antibody Pair #52085 (yellow) and chromogenic staining using PD-L1 (E1L3N) XP® Rabbit mAb #13684.
Unlike many other solutions, SignalStar panels offer amplification. SignalStar technology amplifies the signal from your targets, so that even low levels of expression are readily detectable. See more than you ever have before and detect targets with low levels of expression.
Figure Legend: SignalStar multiplex immunohistochemical analysis of paraffin-embedded human tonsil using PD-1 (Intracellular Domain) (D4W2J) & C0-0008-647 SignalStar Oligo-Antibody Pair #56837 (red) and ProLong Gold Antifade Reagent with DAPI #8961 (blue), compared to PD-1 (Intracellular Domain) (D4W2J) XP® Rabbit mAb #86163 detected with anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 647 Conjugate) #4414, and PD-1 (Intracellular Domain) (D4W2J) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate). All fluorophores have been assigned a pseudocolor, as indicated. Staining was performed on the BOND RX autostainer. Mean fluorescence intensity of PD-1 positive cells was quantified in matched regions of interest from serial tissues sections for the SignalStar assay compared to the indirect and direct fluorescent methodologies.
We validate our antibodies in the SignalStar assay and guarantee them to work as expected. Every SignalStar antibody is validated against the chromogenic gold standard, and is consistently reproducible across replicates.
Our validation process begins with the IHC-P validation, followed by antibody validation for SignalStar Multiplex IHC.
Figure Legend: SignalStar multiplex immunohistochemical analysis of paraffin-embedded human colorectal adenocarcinoma using Pan-Keratin (C11) & C0-0003-488 SignalStar Oligo-Antibody Pair #63566 (green), PD-L1 (E1L3N®) & C0-0005-594 SignalStar Oligo-Antibody Pair #28249 (yellow), CD20 (E7B7T) & C0-0011-647 SignalStar Oligo-Antibody Pair #36775 (red), and CD8ɑ (D8A8Y) & C0-0004-750 SignalStar Oligo-Antibody Pair #62750 (cyan). Representative individual and 4-plex images are included. All fluorophores were assigned a pseudocolor, as indicated. Staining was performed on the BOND RX autostainer. Percent positive cells were quantified in matched regions of interest from serial sections for the SignalStar assay (n=2) compared to the chromogenic.
Stable fluorescent signals with distinct emission peaks make SignalStar panels compatible with your existing imaging instrumentation.
Image your slides using a microscope compatible with channels 488, 594, 647, and 750 nm to process 3-8 plex assays. With a 6-hour protocol run time, it’s possible to stain and image a 4-plex in 1 day or an 8-plex in 2 days. The automated protocol enables overnight staining, greatly reducing hands-on time.
Every antibody used in a SignalStar panel has been independently validated for use in its intended application and guaranteed to perform as expected just like all CST® antibodies—to provide you with reliable, reproducible results, the first time and every time. You’ll also be taking advantage of the most cited antibodies on the market.
Add all your antibodies at once without having to worry about epitope loss or masking.
CST online, easy-to-use SignalStar Multiplex IHC Panel Builder does the design work so you don’t have to.
아래 Cell Signaling Technology의 SignalStar™ Multiplex IHC brochure를 다운로드 해보세요.
