Unsupervised quantification of tissue immunofluorescence in animal models of multiple sclerosis - Instructions for use

Background: In the analysis of animal models of CNS diseases such as experimental autoimmune encephalomyelitis (EAE), immunostaining and histopathology are important readouts. However, the complex morphological features of a tissue staining are often reduced to a single measure which relies on tedious manual planimetry. Furthermore, the measure itself and co-variables such as the region being analysed are chosen in a human decision-making process, which introduces bias. New method: First aim of the present study is to provide an open-source workflow for the high-throughput, unsupervised quantification of different stainings in the spinal cord. We evaluate different EAE models, spinal cord regions and different time points of disease. By applying random forest classification, we compare different measures. Results: Exemplified for glial reactivity, we show that measures and variables interact and that their values are non-normally distributed, hampering the common use of parametric tests. Furthermore, we demonstrate that one-dimensional measures are insufficient descriptors for immunofluorescence data in EAE and thus need to be considered as partly invalid. Comparison with existing methods: We show in a systematic analysis of EAE studies that currently published immunohistological outcomes are highly incompatible regarding methodology and statistics. Furthermore, they lack the report of important information necessary for reproducibility and do not use unsupervised automatic analysis. Conclusions: Our results discover relevant caveats in the currently used methods of immunofluorescence analysis. The provided step-by-step instructions and open-source code are intended to serve as a framework for sensitive, unbiased immunofluorescence analysis of tissue sections in translational research.