Anti human phf tau clone at8

Formalin-fixed hemispheres (4% formaldehyde, 48 h) were embedded in paraffin and sliced on a microtome (4 µm, Leica Biosystems GmbH, Germany). The sections were stained using a BOND-III automated immunostaining system (Leica Biosystems GmbH, Germany). The coronal brain sections (at approximately − 1.6 to − 2.1 mm relative to bregma, 2 mice per line) were stained for Aβ (anti-human Aβ clone 4G8; 1:4000, BioLegend™); TAU (anti-human TAU clone TAU-5, 1:100, Calbiochem); AT8 (anti-human PHF-TAU clone AT8, 1:100, Thermo Scientific); AT180 (anti-human PHF-TAU clone AT180, 1:100, Thermo Scientific); microglia (IBA1, 1:1000, Wako, 019-19741); and astrocytes (GFAP, 1:1000, DAKO, Z033401) as previously described by us [21 (link)–27 (link)]. The Campbell–Switzer (CS) stains were performed as described in Additional file 2: Method S1. After staining, tissue sections were digitized at 230 nm resolution using a Pannoramic slide scanner (3DHistotech, Hungary) and were subject to specialist evaluation by a neuropathologist.

Formalin-fixed sections (3–5 mm-thick) were mounted on slides and deparaffinized. After conducting a routine antigen retrieval protocol, sections were incubated overnight with either a mouse monoclonal antibody against α-synuclein (NCL-L-ASYN; Leica Biosystems) or with a mouse monoclonal antibody anti-human PHF-TAU (clone AT-8; Thermo Scientific). The reaction product was visualized using an automated slide immunostainer (Leica Bond Max) with Bond Polymer Refine Detection (Leica Biosystems Newcastle Ltd).
For semiquantitative analysis LBs density and Lewy neurites (LN) were scored as follows: 0 = absent, + = mild, ++ = moderate, +++ = severe and ++++ = very severe (McKeith et al., 2005 (link)). In the assessment LBs brainstem type and cortical were included. A PD neuropathological stage was assigned according to the staging scheme proposed by Braak. For this measure only the distribution of LBs in any brain region was taken into account instead of the LBs density.