Protocol for immunodetection of α-synuclein pathology in paraffin-embedded liver tissues from murine models of Parkinson's disease

Abstract

The accumulation of α-synuclein (α-Syn) pathology in peripheral tissues of Parkinson's disease (PD) has attracted growing scientific interest in recent years. Here, we present a protocol for the immunodetection of α-Syn pathology in murine liver tissue from models of PD using fluorescence microscopy. We describe steps for liver isolation, fixation, embedding, and immunodetection using an array of antibodies targeting α-Syn. This approach offers valuable applications for studying PD in transgenic mouse models and could be adapted for human liver tissue. For complete details on the use and execution of this protocol, please refer to Hallbeck et al.¹.

Keywords: Cell Biology; Molecular Biology; Neuroscience.

Graphical abstract

Figure 1

The mouse euthanasia system A clear euthanasia chamber connected to wall compressed CO₂ with an exit blocked with a rubber stop placed over a ventilated bench.

Figure 2

Schematic overview of major perfusion steps Schematic representation of a mouse with paws fixed to the bench showing the incision at the mid-belly to expose the thoracic cavity and internal organs followed by perfusion with PBS (Figure generated with BioRender).

Figure 3

Automatic benchtop tissue processor (Leica TP1020)

Figure 4

Components of the embedding station Embedding station with the hot plate on the left and cold plate on the right Note that the metal molds are placed within the chambers covered by black lids (Leica EG1150 Modular Tissue Embedding Station).

Figure 5

Overview of the embedding process Schematic representation of liver placement into the metal mold, the addition of melted paraffin, covering the mold with the cassette lid, and placement on the −20°C embedding surface to allow for solidification (Figure generated with BioRender).

Figure 6

Sectioning station Side and front views of the Microm HM 355S rotary automated paraffin microtome, featuring an integrated cool-cut sample paraffin block holder, a transfer water flow system, and a warm bath with sections floating on its surface.

Figure 7

Antigen retireval system Closed and open side views of the automatic epitope retrieval system (PT Link, pre-Treatment module for tissue specimens).

Figure 8

Immunodetection of α-Syn pathology within the liver of the A30P mouse model of PD Liver tissue sections from the A30P mouse model of PD showing human α-Synuclein inclusions labeled with the aggregate-specific α-Syn antibody MJFR-14-6-2 and Syn-4B12 (panels A–C), and MJFR-14-6-2 and Syn-211 (panels D–F). Scale bars = 50 μm.