Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection

Abstract

Background: The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research. Methods: We used horseradish peroxidase (HRP)-conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event. Results: We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both in cultured cells and in tissue samples. Conclusions: The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.

Proximity ligation assays: a recent addition to the proteomics toolbox

“…The resulting sub-micrometer sized, distinct and bright RCPs are easily distinguishable from potential background fluorescence in an epifluorescence microscope. Alternatively an enzyme-labeled detection oligonucleotide can be used for colorimetric read-out in bright-field microscopy [33]. Since each detected interaction gives rise to one RCP, the detected complexes can be easily enumerated.…”

“…By using the oligonucleotides connected to the antibody as a primer, the RCP will be retained at the site where the proximity probes had bound. An additional advantage of RCA is that it produces single-stranded DNA that can be detected by hybridization of a complementary oligonucleotide carrying a reporter molecule [33].…”

Identification of altered MicroRNA expression patterns in synovial sarcoma

“…An in situ proximity ligation assay (PLA) was employed to clearly and semiquantitatively visualize the expression of miRNA targets in a particular subcellular region (Zieba et al, 2010). Briefly, the HS-SY-II cells grown in Lab-Tek II chamber slides (Thermo Fisher Scientific, Rochester, NY) were incubated for 48 hr with antimiR inhibitors, washed in ice-cold PBS containing 0.1% Triton-X100 for 3 min and fixed with 4% paraformaldehyde for 10 min.…”

Garbage in, garbage out: A critical evaluation of strategies used for validation of immunohistochemical biomarkers

“…It is based on the principle that two or more oligonucleotide‐conjugated antibodies need to bind in close proximity in order to detect a signal, and can be utilized directly in frozen or FFPE tissue sections (Soderberg et al., 2008; Zieba et al., 2010). The binding is visualized by labelling the oligonucleotides with fluorophores or HRP.…”