Getting two birds with one stone: Combining immunohistochemistry and Azan staining in animal morphology


  • Patrick Beckers Institute of evolutionary biology, University of Bonn
  • Müller Claudia Institute of evolutionary biology, University of Bonn
  • Christiane Wallnisch Institute of evolutionary biology, University of Bonn
  • Thomas Bartolomaeus


Animal morphology, Azan staining, correlative approach, Immunohistochemistry, serial sections


Classical histological stained sections have the disadvantage that fine structures, like individual neurites, or specific macromolecules, like neurotransmitters cannot be visualized. Due to its highly specific staining of only one target molecule within the cell, the visualization of delicate structures, which would be superimposed by other tissue layers in classical Azan staining, is possible with immunohistochemistry. However, using immunohistological methods not all tissues of a specimen can be visualized at once. In contrast, density specific stains like Azan allow for a whole staining of the tissues. We provide a step by step protocol of how to combine immunohistochemistry and Azan staining in the same serial paraffin sections. The combination of both methods allows for a highly detailed investigation of structures of interest. The spatial detection of the previous, to Azan staining, gained antibody-labeled signal allows for a much better understanding of animal organ systems. By using serial sections, it is possible to create an aligned image stack that is both Azan stained and also antibody-labeled. Thus enabling a correlative approach that bridges traditional histology with immunohistochemistry in animal morphology.

Author Biography

Patrick Beckers, Institute of evolutionary biology, University of Bonn

Institute of evolutionary biology and ecology


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How to Cite

Beckers P, Claudia M, Wallnisch C, Bartolomaeus T. Getting two birds with one stone: Combining immunohistochemistry and Azan staining in animal morphology. J Biol Methods [Internet]. 2021Sep.2 [cited 2021Dec.4];8(3):e153. Available from: