From fix to fit into the autoptic human brains

  • Beatrice Paradiso | beatrice.paradiso@unipd.it University of Milan, "Lino Rossi" Research Center for the study and prevention of unexpected perinatal death and SIDS Department of Biomedical, Surgical and Dental Sciences; Cardiovascular Pathology Unit, Department of Cardiac, Thoracic and Vascular Sciences, University of Padua Medical School, Padua; Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, Italy. http://orcid.org/0000-0002-4319-4832
  • Michele Simonato University of Ferrara, Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience; School of Medicine, University Vita-Salute San Raffaele, Milan, Italy.
  • Gaetano Thiene University of Padua Medical School, Cardiovascular Pathology Unit, Department of Cardiac, Thoracic and Vascular Sciences, Italy.
  • Anna Lavezzi University of Milan, "Lino Rossi" Research Center for the study and prevention of unexpected perinatal death and SIDS Department of Biomedical, Surgical and Dental Sciences, Italy.

Abstract

Formalin-fixed, paraffinembedded (FFPE) human brain tissues are very often stored in formalin for long time. Formalin fixation reduces immunostaining, and the DNA/RNA extraction from FFPE brain tissue becomes suboptimal. At present, there are different protocols of fixation and several procedures and kits to extract DNA/RNA from paraffin embedding tissue, but a gold standard protocol remains distant. In this study, we analyzed four types of fixation systems and compared histo and immuno-staining. Based on our results, we propose a modified method of combined fixation in formalin and formic acid for the autoptic adult brain to obtain easy, fast, safe and efficient immunolabelling of long-stored FFPE tissue. In particular, we have achieved an improved preservation of cellular morphology and obtained success in postmortem immunostaining for NeuN. This nuclear antigen is an important marker for mapping neurons, for example, to evaluate the histopathology of temporal lobe epilepsy or to draw the topography of cardiorespiratory brainstem nuclei in sudden infant death syndrome (SIDS). However, NeuN staining is frequently faint or lost in postmortem human brain tissues. In addition, we attained Fluoro Jade C staining, a marker of neurodegeneration, and immunofluorescent staining for stem cell antigens in the postnatal human brain, utilizing custom fit fixation procedures.

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Published
2018-08-27
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Issue
Section
Technical Notes
Supporting Agencies
Registry of Cardio-Cerebral-Vascular Pathology, Veneto Region, Italy
Keywords:
Formalin fixation, formic acid, glacial acetic acid, ethanol, post-mortem brain tissue, NeuN, Fluoro Jade C, neural stem cell immunofluorescence staining
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How to Cite
Paradiso, B., Simonato, M., Thiene, G., & Lavezzi, A. (2018). From fix to fit into the autoptic human brains. European Journal of Histochemistry, 62(3). https://doi.org/10.4081/ejh.2018.2944