Immunofluorescent staining is a widespread tool in basic science to understand organ morphology and (patho-) physiology. The analysis of imaging data is often performed manually, limiting throughput and introducing human bias. Quantitative analysis is particularly challenging for organs with complex structure such as the kidney. In this study we present an approach for automatic quantification of fluorescent markers and histochemical stainings in whole organ sections using open source software. We validate our novel method in multiple typical challenges of basic kidney research and demonstrate its general relevance and applicability to other complex solid organs for a variety of different markers and stainings. Our newly developed software tool “AQUISTO”, applied as a standard in primary data analysis, facilitates efficient large scale evaluation of cellular populations in various types of histological samples. Thereby it contributes to the characterization and understanding of (patho-) physiological processes.