Clone at8

For conventional histology, the tissue was fixed in 4% formaldehyde solution for 3–4 weeks, embedded in paraffin and cut in 8 µm thick sections for silver stains and 5 µm for immuno-histochemical techniques. After de-paraffination, sections were stained with Gallyas silver impregnation method. Immunohistochemistry for amyloid-β and hyperphosphorylated tau was performed on an automated immuno-stainer (DAKO Autostainer plus, Glostrup, Denmark) using the following mouse monoclonal primary antibodies: anti- Aβ: clone 6 F/3D (Dako, Glostrup, Denmark; dilution 1/400) and anti- hyperphosphorylated tau protein: clone AT8 (Thermo scientific, Rockford, IL, USA; dilution 1:200). Immunoreaction was visualized using the Envision+ system peroxidase procedures (DAKO) with diaminobenzidine as chromogen.
For label-free Raman spectroscopy, an adjacent tissue block from the same area was fixed in 4% paraformaldehyde in 0.08 M cacodylate buffer (pH 7.3) for maximal one week and then stored in 0.2% paraformaldehyde in 0.08 M cacodylate buffer (pH 7.3) for 5 weeks. It was cut with a vibratome (Leica 1000 S) in slices of 50 µm thickness and stored in PBS for Raman analysis, which was carried out within one week after slicing.

Immunohistochemistry was performed on 4 μm paraffin-embedded sections, targeting the markers of AD pathology pan-Aβ (clone 4G8, BioLegend), Aβ42 (clone 21F12, Elan Pharmaceuticals Ltd) and the phosphorylated (p) tau (clone AT8, ThermoScientific) protein, as well as the microglial motility-related proteins Iba1 (rabbit polyclonal, Wako Chemicals), CFL1 (polyclonal rabbit, ThermoScientific), CORO1A (rabbit polyclonal, LifeSpan Biosciences) and P2RY12 (rabbit polyclonal, Sigma Aldrich). Bound antibodies were visualized using the avidin–biotin–peroxidase complex method (Vectastain Elite, Vector Laboratories) with 3,3′-diaminobenzidine as chromogen and 0.05% hydrogen peroxide as substrate (Vector Laboratories). All sections were counterstained with haematoxylin, then dehydrated and mounted in Pertex (Histolab Products AB). The staining was performed in two batches with each batch containing cases from all groups (Control, AD, iAD). All experiments included a negative control slide incubated in buffer with no primary antibody and a positive control slide containing a specific tissue type known to express the protein of interest (e.g. tonsil).