Envision system peroxidase procedure

Formalin-fixed and paraffin-embedded brain sections of frontal cortex were dewaxed and pretreated with Tris/EDTA buffer pH 9 at high temperature. The following primary antibodies were incubated overnight at 4 °C: polyclonal goat anti-YKL-40 (R&D Systems, AF2599, dilution 1:200), rabbit anti-GFAP (Sigma, G9269, dilution 1:500), phosphorylated tau clone AT8 (Thermo Scientific, MN1020, dilution 1:1000), monoclonal mouse anti-MAP2 (Sigma, M4403, dilution 1:500) and rabbit anti-Iba-1 (Wako Chemicals, 019-19741, 1:500). For IHC, the endogenous peroxidase activity was blocked, sections were HRP-labelled (Dako, Glostrup, Denmark, dilution 1:200) and the reaction was visualized by the EnVision+ system peroxidase procedure (DAKO, Glostrup, Denmark). For IF, sections were incubated for 1 h with Alexa Fluor 488, 555 or 647 (Invitrogen, Carlsbad, CA, USA, dilution 1:1000) secondary antibodies and stained with Sudan black B (Merck, Whitehouse Station, NJ, USA) to mask tissue autofluorescence. Nuclei were stained with Hoechst 33258 (Life Technologies, Carlsbad, CA, USA, dilution 1:1000), and coverslips were added with Immu-Mount (Fisher Scientific, Rockford, USA) mounting medium.

Immunohistochemistry was performed on 5-μm-thick consecutive sections of occipital cortex on an automated stainer (DAKO Autostainer Plus; DAKO, Glostrup, Denmark) using the following primary antibodies: mouse monoclonal anti-amyloid beta clone 6F/3D (dilution 1:400, DAKO, Glostrup, Denmark) and mouse anti-NAB61 (dilution 1:250, a kind gift from Virginia Lee, University of Pennsylvania, Philadelphia, USA). Reaction was visualized by the EnVision + system peroxidase procedure (DAKO, Glostrup, Denmark). Full-section scans were obtained with Pannoramic MIDI II (3DHistech, Budapest, Hungary) using a 40x objective. Cortical grey matter of each case was manually delimitated with pannoramic viewer software (3DHistech, Budapest, Hungary). An adaptation of the algorithm previously described^{64 (link)} was developed to quantify total Aβ and NAB61 immunoreactivity with MATLAB software (The Math Works, Inc., Natick, MA, USA). Specifically, a colour deconvolution was used to split the channels and a mean local filter was then applied to segment the structures (Supplementary Fig. S1).

We assessed formalin-fixed and paraffin-embedded tissue blocks from frontal cortex Brodmann areas 8/9. Immunohistochemistry was performed on 5-μm-thick sections on an automated stainer (DAKO Autostainer Plus; DAKO, Glostrup, Denmark) using the following primary antibodies: anti-amyloid β (clone 6F/3D, dilution 1:400; DAKO) and anti-phosphorylated tau (clone AT8, dilution 1:2000; Thermo Scientific, Waltham, MA). Reaction was visualized by the EnVision+ system peroxidase procedure (DAKO).
Immunoreactive structures of AT8 (NFTs, NTs, pretangles, dystrophic neurites, balloon cells, Pick bodies, ramified astrocytes, astrocytic plaques, tufted astrocytes and coiled bodies) and β-amyloid (mature, primitive and diffuse plaques) were systematically assessed in all cases. Neurofibrillary pathology was staged according to Braak criteria [25 , 26 (link)]. β-amyloid phases were evaluated according to Thal criteria [27 (link)]. The National Institute on Aging-Alzheimer’s Association Guidelines for neuropathologic assessment of AD was also applied [24 ]. Healthy controls without tau or amyloid pathology were included. Neuropathologic evaluation was carried out by three investigators on a multiheaded microscope.