Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with peroxidase detection.
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.
Zieba
Agata
A
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
Wählby
Carolina
C
Hjelm
Fredrik
F
Jordan
Lee
L
Berg
Jonathan
J
Landegren
Ulf
U
Pardali
Katerina
K
D000818:Animals; D002478:Cells, Cultured; D004958:Estradiol / Q000032:analysis; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis; D011959:Receptors, Estradiol / Q000032:analysis; D051899:Smad2 Protein / Q000032:analysis
D004958:Estradiol; D006735:Horseradish Peroxidase; D011506:Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011959:Receptors, Estradiol; D051899:Smad2 Protein