Self-crosslinkable protein hydrogel stamps for rapid and wash-free immunostaining in cells and tissues

Immunostaining is essential for cancer biomarker detection, such as HER2 and EGFR, but conventional methods often require prolonged incubation and multiple washing steps. Here, we developed self-crosslinkable protein hydrogel (SPH) stamps for simple, rapid, and reusable immunostaining of cells and tissues. Mixing SpyTag-fused lumazine synthase protein nanoparticles (AaLS-ST) with SpyCatcher tandem dimers (SC-SC) at a 2:1 molar ratio formed stable, self-crosslinked hydrogels with hydrophilic pores and high mechanical strength. Flat-disc SPH stamps, mounted on plastic bars, efficiently absorbed antibody solutions and transferred them to target biomarkers via stamping. HER2-overexpressing SKBR-3 and EGFR-overexpressing MDA-MB-468 cells were specifically stained with PE-conjugated anti-HER2 antibody (aHER2-Ab-PE) and APC-conjugated anti-EGFR antibody (aEGFR-Ab-APC), respectively, within 10 min without washing through simple stamping. A single SPH stamp loaded with multiple antibodies selectively stained the corresponding cells without washing steps, while sequential stamping of primary and secondary antibodies enabled simplified two-step immunostaining. Reusability was validated through repeated staining of multiple fixed cell slides and tumor tissue slices with a single antibody loading. SPH stamps provide a rapid, versatile, and reusable platform for immunostaining of cells and tissues, providing a promising alternative to conventional methods. STATEMENT OF SIGNIFICANCE: Immunostaining is central to cancer diagnostics but limited by lengthy incubation and multiple washing steps. Self-crosslinkable protein hydrogel (SPH) stamps are developed, which rapidly absorb and release antibodies, enabling target-specific staining of cells and tissues within minutes without washing. SPH stamps can be reused across multiple samples with a single antibody loading, including tissue sections. They also enable selective staining of corresponding cells with a single loading of multiple antibodies without washing steps, as well as simplified two-step immunostaining using sequential primary and secondary antibody stamping. This platform integrates speed, simplicity, and reusability, offering a promising protein-based alternative for cell and tissue immunostaining with potential impact in diagnostic pathology and high-throughput analysis.