Quantitative image analysis of protein expression and colocalisation in skin sections

Abstract

Immunofluorescence (IF) and in situ proximity ligation assay (isPLA) are techniques that are used for in situ protein expression and colocalisation analysis, respectively.However, an efficient quantitative method to analyse both IF and isPLA staining on skin sections is lacking. Therefore, we developed a new method for semi-automatic quantitative layer-by-layer measurement of protein expression and colocalisation in skin sections using the free open-source software CellProfiler. As a proof of principle, IF and isPLA of ichthyosis-related proteins TGm-1 and SDR9C7 were examined. The results indicate that this new method can be used for protein expression and colocalisation analysis in skin sections. [1] isPLA signals, each representing one colocalisation, are seen as fluorescent spots under the microscope.[1]Both IF and isPLA have already been applied in subjective and semi-quantitative analysis of protein expression and colocalisation in skin sections. [2,3] However, an efficient quantitative image analysis method for IF and isPLA is lacking. A previous report described advanced automatic segmentation of the epidermal part of the images using the commercial scripting tool Matlab. [4] Another study used the open-source software ImageJ to quantify colorimetric staining of melanin in skin without segmentation of the epidermis. [5] CellProfiler is also a free and open-source software and does not require any programming skills, which makes it easy to handle. | QUESTION ADDRESSEDWe developed a new, efficient method for objective quantitative image analysis of protein expression and colocalisation in different epidermal layers of skin sections. | EXPERIMENTAL DESIGN | Human skin samplesThis study was conducted in conformity with Declaration of Helsinki Principles and approved by Regional Ethical Review Board at Uppsala University. After informed and written consent and local anaesthesia, punch biopsies (3 mm) were obtained from healthy skin donors, fixed in 4% buffered formalin and embedded in paraffin. | 197 METHODS LETTERS TO THE EDITOR | Immunofluorescence (IF) and in situ proximity ligation assay (isPLA)IF staining was performed as described in Supporting Information. The isPLA was performed according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). Microscopy images of IF and isPLA staining were acquired as described in Supporting Information.Images for IF were collected in single focal plane, whereas isPLA images were collected as z-stacks to enable detection of signals present at different depths in the skin sections. | Quantitative analysis of IF staining and isPLA signalsA detailed instruction on how to use the CellProfiler pipelines, Pipeline I projection, Pipeline II region-of-interest (ROI) and Pipeline III measurement, can be found in the Supporting Information. The CellProfiler software and the pipelines used in this study will be available for downloading at www.cellprofiler.org.

Patients with congenital ichthyosis and TGM1 mutations overexpress other ARCI genes in the skin: Part of a barrier repair response?

“…Some genes showing pertinent changes at the transcriptional level were also analysed at the protein level by immunofluorescence (IF) staining of skin sections (Figure A and B). By applying image analysis, protein expression—represented by median fluorescence intensity—was also analysed in different epidermal layers using recently developed pipelines for the CellProfiler software (Figure C). It can be seen that the expression of CYP4F22, FATP4 (encoded by SLC27A4 ) and CerS3 in patient skin is markedly increased in the upper spinous and granular layers and also extends to additional epidermal layers compared to control skin (Figure A‐C).…”

Ichthyosis: A Road Model for Skin Research

“…The co-localization of 2 other ARCI proteins, TGm-1 and SDR9C7, was studied in more detail in healthy control skin using in situ proximity ligation assay (isPLA), which generates a signal when 2 different proteins are at a distance of less than 30 nm from each other (50). While filaggrin did not produce any isPLA signals with either of the 2 ARCI proteins, together they produced a strong signal in stratum granulosum consistent with a close interaction between TGm-1 and SDR9C7 in a chain of events leading to a proper formation of CLE (51). TGm-1 has also been found to co-localize with 12R-LOX and eLOX-3 in stratum granulosum of normal epidermis, but not in ARCI epidermis with inactivating mutations in NIPAL4 (encoding ichthyin) (52).…”

Acylceramide is a key player in skin barrier function: insight into the molecular mechanisms of skin barrier formation and ichthyosis pathogenesis

“…Mutations in SDR9C7 are known to cause ARCI [74][75][76]. SDR9C7 codes short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) [77,78], which is predominantly present in the epidermis [79]. An ARCI patient with an SDR9C7 mutation and Sdr9c7 KO mice showed significantly reduced amounts of ceramides bound to CCE proteins [14].…”