title: Trypan Blue staining method for quenching the autofluorescence of RPE cells for improving protein expression analysis.
abstract: Retinal pigment epithelial (RPE) cells are currently in the "spotlight" of cell therapy approaches to some retinal diseases. The analysis of the expressed proteins of RPE primary cells is an essential step for many of these approaches. But the emission of autofluorescence by RPE cells produces higher background noise interference thereby creating an impediment to this analysis. Trypan Blue (TB), a routinely used counterstain, has the capacity to quench this autofluorescence, if it is used in optimized concentration. The results from the method developed in our study indicate that incubation of the cultured RPE cells with 20 μg/ml of TB after immunolabelling (post-treatment) as well as incubation of the retinal tissue specimens with same concentration before paraffin embedding, sectioning and immunolabelling (pre-treatment) can be applied to effectively quench the autofluorescence of RPE cells. Thus it can facilitate the evaluation of expressed cellular proteins in experimental as well as in pathological conditions, fulfilling the current requirement for developing a method which can serve to eliminate the autofluorescence of the cells, not only in cell cultures but also in tissues samples. This method should significantly increase the quality and value of RPE cell protein analysis, as well as other cell protein analysis performed by Flow cytometry (FC) and Immunohistochemistry (IHC) techniques.
authors.affiliation: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain. girish@ioba.med.uva.es
authors.forename: Girish K
authors.initials: GK
authors.lastname: Srivastava
authors.forename: Roberto
authors.initials: R
authors.lastname: Reinoso
authors.forename: Amar K
authors.initials: AK
authors.lastname: Singh
authors.forename: Ivan
authors.initials: I
authors.lastname: Fernandez-Bueno
authors.forename: Denise
authors.initials: D
authors.lastname: Hileeto
authors.forename: Mario
authors.initials: M
authors.lastname: Martino
authors.forename: Maria T
authors.initials: MT
authors.lastname: Garcia-Gutierrez
authors.forename: Jose M Pigazo
authors.initials: JM
authors.lastname: Merino
authors.forename: Nieves Fernández
authors.initials: NF
authors.lastname: Alonso
authors.forename: Alfredo
authors.initials: A
authors.lastname: Corell
authors.forename: J Carlos
authors.initials: JC
authors.lastname: Pastor
mesh_terms: D000818:Animals; D016477:Artifacts; D002478:Cells, Cultured; D004396:Coloring Agents; D004847:Epithelial Cells / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D016612:Paraffin Embedding; D015203:Reproducibility of Results; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D013552:Swine; D014343:Trypan Blue
chemical_list: D004396:Coloring Agents; D005136:Eye Proteins; D014343:Trypan Blue