- #CELLPROFILER SAVING IMAGES HOW TO#
- #CELLPROFILER SAVING IMAGES SKIN#
- #CELLPROFILER SAVING IMAGES SOFTWARE#
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#CELLPROFILER SAVING IMAGES SKIN#
(D) The number of isPLA signals/1000 μm 2 for each layer of isPLA of TGm-1/SDR9C7, TGm-1/filaggrin (biological negative control) and technical negative control with only one primary antibody (Ab) and nuclei DAPI signal was plotted against skin depth. isPLA signals were outlined in white, indicating that they were detected and counted by Pipeline III. (C) The ROI was automatically divided into layers, each corresponding to 8 μm in thickness (almost one layer of viable keratinocytes), by Pipeline III. (B) The region-of-interest (ROI) was manually outlined using Pipeline II. (A) Projected image of isPLA of TGm-1/SDR9C7 (red) with nuclear counterstaining (blue) using Pipeline I. Quantification of isPLA colocalisation of TGm-1 and SDR9C7 using customised CellProfiler pipelines.
The pipelines can also be used for analyses of proteins expressed in the basal epidermal layers (see Supporting Information and Fig. The median fluorescence intensities for TGm-1 and SDR9C7 reveal maximum expression in upper stratum granulosum (Figure 1G). 7 ROIs were manually outlined with Pipeline II (Figure 1B,E) and subsequently automatically divided into up to 20 layers by Pipeline III (Figure 1C,F). Figure 1 shows an example of IF staining of transglutaminase-1 (TGm-1) (Figure 1A) and short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) (Figure 1D), 2 genes causing autosomal recessive congenital ichthyosis. The 3 pipelines were used to measure IF staining intensities (Figure 1) and isPLA signals (Figure 2) in skin sections.
#CELLPROFILER SAVING IMAGES SOFTWARE#
The CellProfiler software and the pipelines used in this study will be available for downloading at 4 RESULTS AND DISCUSSION
#CELLPROFILER SAVING IMAGES HOW TO#
3.3 Quantitative analysis of IF staining and isPLA signalsĪ 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. 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. Microscopy images of IF and isPLA staining were acquired as described in Supporting Information. The isPLA was performed according to the manufacturer's instructions (Sigma-Aldrich, St. IF staining was performed as described in Supporting Information. 3.2 Immunofluorescence (IF) and in situ proximity ligation assay ( isPLA)
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. This study was conducted in conformity with Declaration of Helsinki Principles and approved by Regional Ethical Review Board at Uppsala University. 3 EXPERIMENTAL DESIGN 3.1 Human skin samples We developed a new, efficient method for objective quantitative image analysis of protein expression and colocalisation in different epidermal layers of skin sections. Therefore, we developed new CellProfiler pipelines for analysis of IF and isPLA staining of skin sections in an objective and quantitative way. 6 CellProfiler uses so-called pipelines, which are composed of individual image-processing modules, to analyse many images automatically.
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5 CellProfiler is also a free and open-source software and does not require any programming skills, which makes it easy to handle. 4 Another study used the open-source software ImageJ to quantify colorimetric staining of melanin in skin without segmentation of the epidermis. A previous report described advanced automatic segmentation of the epidermal part of the images using the commercial scripting tool Matlab. 2, 3 However, an efficient quantitative image analysis method for IF and isPLA is lacking. 1īoth IF and isPLA have already been applied in subjective and semi-quantitative analysis of protein expression and colocalisation in skin sections. 1 isPLA signals, each representing one colocalisation, are seen as fluorescent spots under the microscope.
The isPLA detects colocalisation of 2 target proteins at a maximal distance of 30 nm in situ with high specificity. IF staining can reveal the location and relative expression level of a protein. Immunofluorescence (IF) and in situ proximity ligation assay ( isPLA) are methods used for studies of protein expression and colocalisation, respectively.
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