CFP™ Spatial Biology Platforms

Core Technology: Cleavable Fluorescent Probe (CFP™)

Similar to tyramide signal amplification (TSA), CFP™ cleavable TSA fluorophores leverage the catalytic activity of horseradish peroxidase (HRP) to enable the covalent deposition and binding of labeled fluorophores onto target proteins or nucleic acid sequences in situ. These fluorophores are engineered for much higher reactivity than tyramide fluorophores, making the imaging process significantly faster and more robust than conventional TSA-based applications.

Unlike conventional TSA fluorophores, CFP™ fluorescent signals can be cleaved using mild cleavage reagents, enabling ultra-sensitive reiterative staining with standard antibodies. Furthermore, CFP™ fluorophore technology is highly versatile and can be seamlessly integrated into protocols requiring HRP-based detection, including immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), in situ hybridization (ISH), and proximity ligation assays (PLA).

Figure 1. Mechanism of Action for Cleavable TSA Fluorophores

CFP™ Multiplexing Strategy

The multiplexing strategy based on the CFP™ probe allows iterative cycles of staining, imaging, and fluorophore cleaving. In each cycle, protein targets are first recognized by primary antibodies from different species and of varied immunoglobulin classes. Subsequently, the first target is stained by secondary antibodies and the CFT™ cleavable fluorophore. HRP catalyzes the coupling reaction between CFT and the tyrosine residues on the endogenous proteins in close proximity. After imaging, the fluorophores are gently cleaved with nearly 100% efficiency and HRP is simultaneously deactivated. Through reiterative analysis cycles, many distinct proteins can be quantitatively profiled in single cells in-situ.

Key Features

• Ultra-High Multiplexing Capability: Capable of detecting dozens to potentially hundreds of protein biomarkers on a single slide, overcoming the spectral limitations of traditional immunohistochemistry (IHC) or immunofluorescence (IF).
• Exceptional Tissue Preservation: The chemical cleavage process is highly efficient and extremely gentle. It preserves tissue morphology, RNA integrity, and protein antigenicity, allowing for downstream assays like H&E staining on the same slide.
• Broad Sample Compatibility: Fully compatible with both formalin-fixed paraffin-embedded (FFPE) and fresh frozen tissue sections.
• Automation Friendly: The CFP™ assay is automation friendly. There are both a manual version and a fully automated version, accessible through S-CORE™ Auto-Stainer.
• High Resolution and Sensitivity: Provides true single-cell and sub-cellular resolution with high signal-to-noise ratios, ensuring accurate cell phenotyping and spatial localization.

CFP™ Fluorophore Packs

Amerigo Scientific offers CFP™ Fluorophore Packs and each pack contains CFP™ cleavable TSA fluorophore and DMSO for reconstitution.

CFP™ Multiplex Protein Detection Kits

The CFP™ Multiplex Protein Detection Kits are advanced, highly scalable spatial biology reagent solutions designed for multiplexed spatial proteomics. These kits enable researchers to visualize, identify, and quantify dozens of protein targets simultaneously on a single tissue section while preserving the native tissue architecture.

Applications

The CFP™ Multiplex Protein Detection Kits are highly valuable for complex biological research, including:

• Tumor Microenvironment (TME) Profiling: Mapping the spatial distribution of cancer cells, immune cells, and stroma.
• Immune Phenotyping: Deep profiling of immune cell subsets and their activation/exhaustion states in situ.
• Spatial Biomarker Discovery: Identifying novel spatial signatures for drug development, companion diagnostics, and personalized medicine.
• Neuroscience & Tissue Atlas Construction: Mapping complex cellular networks in the brain and other organs.

S-CORE™ Auto-Stainer

S-CORE™ Auto-Stainers are designed as an "open-source" automated staining platform dedicated to spatial biology protocols. It provides a highly flexible, affordable, and fully customizable solution for laboratories conducting multiplexed spatial omics assays. The system is built to automate essential staining steps for various applications, including H&E, IHC, IF, and RNA detection.

S-CORE™ Specifications

Applications

Case 1: Multiplex Immunofluorescence (mIF) for High-Plex Spatial Protein Profiling

CFP™ technology enables researchers to perform ultra-high-plex protein profiling on a single tissue section, providing deeper spatial insights than conventional IHC panels. As shown in Figure 2, researchers successfully performed multi-round CFP staining to detect 12 different protein markers on the exact same slide of an FFPE human LUSC tissue. This high-plex spatial profiling reveals a highly actionable and detailed immune context within the tumor microenvironment

Figure 2. Multi-rounds CFP staining of protein markers on the same slide FFPE human lung squamous cell carcinoma (Scale bars =100 μm).

Case 2: Multiomics Co-Detection

CFP™ technology can be seamlessly combined with advanced RNA ISH and spatial transcriptomics assays, offering a comprehensive multimodal approach to unveil complex biological processes. This is particularly valuable for studying targets that lack specific antibodies (such as secreted molecules or non-coding RNA) and for orthogonal biomarker cross-validation. As shown in Figure 3, a multi-round 20-plex assay was performed on a single FFPE human tonsil tissue section, simultaneously detecting 19 different protein markers and 1 RNA marker on the exact same slide.

Figure 3. Multi-round 20-plex CFP staining on the same FFPE human tonsil tissue section (Brightness adjusted for visualization. Scale bars, 200 μm).