From fix to fit into the autoptic human brains

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.

Figure 1.

Human dentate gyrus in post-mortem cases. Hematoxylin and eosin staining (HE), magnification 20x and automatized NeuN immunohistochemistry performed in four different fixation protocols obtained from the same autoptic case, magnification 4x. A) HE in formalin fixation (classic protocol); B) HE in formalin fixation and formic acid treatment; C) HE in liquid fixative (LF) based on glacial acetic acid and formalin solution; D) HE in LF and formic acid treatment; E) NeuN staining in A protocol; F) NeuN staining in B protocol; G) NeuN staining in C protocol; H) NeuN staining in D protocol. HE staining shows that by B protocol the tissue matrix is more compact, without empty and irregular artefactual spaces, nuclei and nucleoli are more visible and better preserved and cell shape appears well represented (B). Automatized staining for NeuN results surely positive with little background exclusively in B protocol (F). Automatized NeuN immunostaining in fixation A protocol shows high level of background and paler staining for CA4 neurons (black arrow heads) and more loose parenchyma (E), compared to automatized NeuN staining in B protocol (F).

Figure 2.

Human dentate gyrus in post-mortemcases. Automatized immunohistochemistry performed against NeuN, GFAP and NF brain antigen markers, magnification 20x (A-D) and manual staining against NeuN antigen, magnification 2.5x (E-H). A) Automatized NeuN immunostaining in A-type fixation protocol appears paler with more background, false positivity for glia and astrocytes (white arrow heads) and negativity of CA4 area neurons (black arrow heads), compared with B. B) Automatized NeuN immunostaining in B-type fixation protocol. C) Automatized GFAP immunostaining in B-type fixation protocol. D) Automatized NF immunostaining in B-type fixation protocol. E-H) Human Hippocampus in long-term fixation of the same post-mortem case. Manual immunostaining in A-type fixation protocol, with different antibody concentration respectively in E,G) and F,H).

Figure 3.

Human adult Fluoro Jade C (FJC) staining of hippocampal neurodegeneration, magnification 20x. A) Monolateral temporal post-traumatic epilepsy with left mesial temporal sclerosis (MTS), FJC in green; B) Controlateral temporal lobe in absence of neurodegeneration, FJC in green; C) DAPI in blue of left MTS; D) DAPI in blue of contralateral side. E, F) merged view of A-C and B-D.

Figure 4.

Human postnatal dentate gyrus in SIDS case. Nestin immunostaining, magnification 5x and Fluoro Jade C (FJC) neurodegeneration detection, magnification 20x. A) Nestin immunostaining in LF protocol: nestin- positive neural precursors are migrating from the subgranular (SGZ) and subventricular zone (SVZ); B) Human postnatal FJC staining (in green) of CA3 area of hippocampus in SIDS case, without any aspect of neurodegeneration, compared with CA3 area of perinatal FJC positive hypoxic ischemic encephalopathy in the control case (C), nuclei stained in blue by DAPI.

Figure 5.

Human postnatal neurogenesis in subventricular zone (SVZ) by antigen of stemness in SIDS case. Immunofluorescence staining in LF fixation protocol, magnification 20x. A) Nuclei in blue, DAPI; B) SOX2 in red; C) Nestin in green; D) merged image of A, B, C; E) Human postnatal neurogenesis in subventricular zone (SVZ) in SIDS case. Merged image of immunofluorescence staining: nuclei in blue, DAPI; nestin in green; SOX2 in red, in LF fixation protocol, magnification 40x.