Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

Cells. 2020 Apr 1;9(4):852. doi: 10.3390/cells9040852.


The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.

Keywords: immunofluorescence; immunohistochemistry; proteomics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / metabolism
  • Biotin / chemistry
  • Fluorescence
  • Fluorescent Dyes / chemistry
  • Formaldehyde / chemistry
  • HeLa Cells
  • Histones / metabolism
  • Humans
  • Ki-67 Antigen / metabolism
  • Lysine / metabolism
  • Methylation
  • Paraffin Embedding
  • Proteins / analysis*
  • Streptavidin / chemistry*
  • Tissue Fixation


  • Antibodies
  • Fluorescent Dyes
  • Histones
  • Ki-67 Antigen
  • Proteins
  • Formaldehyde
  • Biotin
  • Streptavidin
  • Lysine