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Evaluating real-time immunohistochemistry on multiple tissue samples, multiple targets and multiple antibody labeling methods
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.ORCID-id: 0000-0001-9141-9242
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för radiologi, onkologi och strålningsvetenskap, Enheten för biomedicinsk strålningsvetenskap.
2013 (Engelska)Ingår i: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 6, s. 542-Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Background

Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method.

Results

Three different antibodies labeled with fluorescent or radioactive labels were applied on nine different tissue samples from either human or mouse, and the results for all RT-IHC analyses distinctly show that the method is generally applicable. The collected binding curves showed that the majority of the antibody-antigen interactions did not reach equilibrium within 3 hours, suggesting that standardized protocols for immunohistochemistry are sometimes inadequately optimized. The impact of tissue size and thickness as well as the position of the section on the glass petri dish was assessed in order for practical details to be further elucidated for this emerging technique. Size and location was found to affect signal magnitude to a larger extent than thickness, but the signal from all measurements were still sufficient to trace the curvature. The curvature, representing the kinetics of the interaction, was independent of thickness, size and position and may be a promising parameter for the evaluation of e.g. biopsy sections of different sizes.

Conclusions

It was found that RT-IHC can be used for the evaluation of a number of different antibodies and tissue types, rendering it a general method. We believe that by following interactions over time during the development of conventional IHC assays, it becomes possible to better understand the different processes applied in conventional IHC, leading to optimized assay protocols with improved sensitivity.

Ort, förlag, år, upplaga, sidor
2013. Vol. 6, s. 542-
Nationell ämneskategori
Annan medicinsk grundvetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-223781DOI: 10.1186/1756-0500-6-542OAI: oai:DiVA.org:uu-223781DiVA, id: diva2:714053
Tillgänglig från: 2014-04-25 Skapad: 2014-04-25 Senast uppdaterad: 2018-01-11Bibliografiskt granskad

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Dubois, LouiseAndersson, KarlAsplund, AnnaBjörkelund, Hanna

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Enheten för biomedicinsk strålningsvetenskapInstitutionen för immunologi, genetik och patologiScience for Life Laboratory, SciLifeLab
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