Antibody Validation for SignalStar^™ Multiplex IHC

SignalStar Multiplex IHC is an advanced technology for labeling multiple cellular proteins simultaneously in FFPE tissue samples using specific primary antibodies and fluorescent detection reagents. It offers high accuracy and reliability in visualizing and analyzing protein expression in tissue samples.

Because CST validates all of our antibodies in each application independently, SignalStar Multiplex IHC antibodies are only recommended when we have validated them specifically for use with SignalStar technology. That means that you can be confident that your SignalStar antibody will work as expected in your assay every time, guaranteed.

We provide expert support to optimize the application of SignalStar Multiplex IHC technology to reduce the time in your research. Our team is available to answer questions and offer technical assistance.

Validation Steps:

• All SignalStar antibodies must pass a rigorous IHC-P validation process before SignalStar validation is performed.
• SignalStar technology is directly compared to traditional DAB chromogenic labeling of serial sections to confirm specificity.
• SignalStar oligo-conjugated antibodies are optimized across numerous multiplexed panel variations, in all available fluorescent channels and imaging rounds.
• SignalStar assays are validated across unique replicates, instrumentation, days, and automated versus manual protocols.
• Chromogenic staining using SignalStar oligo-conjugated antibodies is used to confirm antigen specificity is maintained throughout the conjugation process.
• Antibodies are tested on positive control tissues known to express targets of interest.
• Subcellular localization and co-localization of targets are considered.
• SignalStar oligo-conjugated antibody performance is assessed in relevant models of cancer, with human cancer tissue arrays used to demonstrate antibody performance over a broad spectrum of tissue types.
• Thorough lot testing ensures the reproducibility necessary for accurate SignalStar results. Dilutions and protocols are predetermined and specified.

Chromogenic Comparison

SignalStar multiplex immunohistochemical analysis of paraffin-embedded human gastric adenocarcinoma, using Pan-Keratin (C11) & C0-0003-488 SignalStar Oligo-Antibody Pair #63566 (top left, green), PD-L1 (E1L3N) & C0-0005-594 SignalStar Oligo-Antibody Pair #28249 (upper middle left, yellow), CD20 (E7B7T) & C0-0011-647 SignalStar Oligo-Antibody Pair #36775 (lower middle left, red), and CD8α (D8A8Y) & C0-0004-750 SignalStar Oligo-Antibody Pair #62750 (bottom left, cyan), compared to chromogenic immunohistochemical analysis of serial sections of paraffin-embedded human gastric adenocarcinoma using Pan-Keratin (C11) Mouse mAb #4545 (top right), PD-L1 (E1L3N) XP® Rabbit mAb #13684 (upper middle right), CD20 (E7B7T) XP® Rabbit mAb #48750 (lower middle right), and CD8α (D8A8Y) Rabbit mAb #85336 (bottom right). All fluorophores have been assigned a pseudocolor, as indicated. All protocols were performed on the BOND RX Fully Automated Research Stainer by Leica Biosystems. Percent positive cells were quantified in matched regions of interest from serial sections for the SignalStar assay (n=2) compared to the chromogenic.

Conjugate Optimization

A) SignalStar immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma, using CD3ε (D7A6E) & C0-0001- 488 SignalStar Oligo-Antibody Pair #92856 (top left, green), CD3ε (D7A6E) & C0-0001-594 SignalStar Oligo-Antibody Pair #84634 (top right, yellow), CD3ε (D7A6E) & C0-0001-647 SignalStar Oligo-Antibody Pair #33888 (bottom left, red), CD3ε (D7A6E) & C0-0001-750 SignalStar Oligo-Antibody Pair #51754 (bottom right, cyan). All fluorophores have been assigned a pseudocolor, as indicated. Staining was performed on the BOND RX autostainer. B) SignalStar immunohistochemical analysis in serial sections of paraffin-embedded human infiltrating ductal carcinoma of the breast, the first imaging round (left) compared to the second imaging round (right), using Vimentin (D21H3) CO-0012-750 SignalStar Oligo-Antibody Pair #61002 (top panel, cyan) or HER2/ErbB2 (D8F12) & CO-0019-488 SignalStar Oligo-Antibody Pair #31149 (bottom panel, green). All fluorophores have been assigned a pseudocolor, as indicated. Staining was performed on the BOND RX autostainer.

Protocol Validation

SignalStar immunohistochemical analysis in serial sections of paraffin-embedded human colorectal adenocarcinoma, with staining performed using the manual protocol (left) compared to the Bond RX protocol (right), using Granzyme B (D6E9W) & CO-0009-647 SignalStar Oligo-Antibody Pair #37369 (magenta) or Pan-Keratin (C11) & C0-0003-647 SignalStar Oligo-Antibody Pair #16373 (red). All fluorophores have been assigned a pseudocolor, as indicated.