Quantifying Epidermal Protein Expression

Image acquisition of HEE immunostainings was performed by a ZEISS Axio Imager equipped with a ZEISS Axiocam 105 color Digital Camera and a 40× objective. The ZEISS Axiocam 105 color is a compact 5-megapixel camera (2560 × 1920 pixels) for high resolution images with a 1/2.5” sensor. Two images per slide were chosen as representative for the whole culture and stored in CZI format. The images were analyzed with the cell image analysis software CellProfiler (Broad Institute) [64 (link)]. Software pipelines for filaggrin (FLG), loricrin (LOR), and involucrin (IVL) analysis were created (available upon request) and GraphPad Prism 9.0 was used for the visualization of the data. The quantified data is shown as the protein area occupied (µm²) per millimeter length of the epidermis. Because this value does not normalize for the thickness of the epidermis (differences in epidermal thickness were observed after compound stimulation), we also calculated the area of expression as percentage of the epidermal surface for comparison.

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Rikken G., van den Brink N.J., van Vlijmen-Willems I.M., van Erp P.E., Pettersson L., Smits J.P, & van den Bogaard E.H. (2022). Carboxamide Derivatives Are Potential Therapeutic AHR Ligands for Restoring IL-4 Mediated Repression of Epidermal Differentiation Proteins. International Journal of Molecular Sciences, 23(3), 1773.

Publication 2022

Axio Imager Axiocam 105 GraphPad Prism 9.0

Camera Cell Epidermal Filaggrin Involucrin Loricrin Prism Protein

Corresponding Organization : Radboud University Nijmegen

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Variable analysis

independent variables

Stimulation with compounds

dependent variables

Protein area occupied (µm^2) per millimeter length of the epidermis
Area of expression as percentage of the epidermal surface

control variables

Imaging equipment (ZEISS Axio Imager with ZEISS Axiocam 105 color Digital Camera and 40× objective)
Image analysis software (CellProfiler)
Data visualization software (GraphPad Prism 9.0)
Image format (CZI)

controls

No explicit mention of positive or negative controls

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