Smi31

Eyes were preserved in Davidson fixative and paraffin embedded overnight. Six μm sections were made through the optic nerve, deparaffinized and stained with hematoxylin and eosin (H&E). For immunofluorescence on sections, mice were anesthetized and transcardially perfused with 4% paraformaldehyde. Eyes and optic nerves were collected and cryoprotected with 30% sucrose. The tissue was embedded in Tissue Tek OCT compound followed by freezing in an isopentane bath cooled by liquid nitrogen. To reduce non-specific binding, 10–40 μm thick sections were blocked with 10% normal donkey serum for 1 h followed by staining for CD11b (BDPharmingen), CD11c-GFP (Invitrogen), Ki67 (Abcam), SMI-31 (Covance) and IsolectinB₄ (Invitrogen). Sections were incubated as described [54 (link), 62 (link)] with antibodies overnight at 4 °C. After three rinses in PBS, appropriate secondary antibodies were applied (Alexa Fluor 350, 488 and 594; Molecular Probes, CA). The tissues were incubated at RT in the dark for 3 h and counterstained with DAPI. Primary antibody was omitted to confirm specificity. Fluorescence images were obtained using a confocal scanning laser microscope (Olympus Fluoview 1000) or a LEICA DM 4000B fluorescence microscope.

For histochemical or immunohistochemical staining, every 10th coronal 6 μm section was cut from the center of the lesion (bregma −1 mm to +1 mm), and a total of 5 paraffin sections were used. Histochemical staining for BS⁺ and LFB⁺, and immunohisto-staining for Syn⁺ (Chemicon, Temecula, CA, USA) and SMI31 (Covance, Princeton, NJ, USA) single staining were performed. Double-immunohisto staining for nestin (Santa Cruz, Santa Cruz, CA, USA) and Sox2 (Santa Cruz), and 4’,6-diamido-2-phenylindole (DAPI, Santa Cruz) were employed.
The Golgi-Cox impregnation method along with the FD Rapid Golgi Stain kit (FD Neuro-Technologies, Columbia, MD, USA) were used to identify Golgi-Cox impregnated dendrites and spines, according to kit instructions [45 (link)].

The following antibodies were used: actin (Sigma A3853), ACO2 (Sigma, HPA001097), APP (Chemicon MAB348), ATF6 (Abcam 40256), CAII (Said Ghandour), GFAP (Chemicon MAB3402), Iba1 (Wako 019-19741), MAC3 (Pharmigen 01781D), NF200 (Sigma N4142), Olig2 (Charles Stiles/John Alberta, DF308), OXCT1 (Proteintech Europe 12175-1AP), SMI31 (Covance SMI-31P), VDAC (Rockland), isolectin IB4 coupled to Alexa 594 (Vectorlab). For generation of MCT1 (SLC16a1) antisera, rabbits were immunized with the intracellular peptide 221–236 of mouse MCT1 (CDANTDLIGGSPKGEKL). Anti-MCT1 antibodies were purified by affinity chromatography.

Paraffin‐embedded sections were double‐stained as previously described 6. Primary antibodies used were rabbit anti‐Calbindin‐D28K (1:500, Sigma‐Aldrich, Dorset, UK) for Purkinje cell identification; mouse monoclonal antibody to phosphorylated neurofilaments SMI‐31 (1:500, Covance, New Jersey, USA); mouse monoclonal anti‐hyperphosphorylated neurofilament SMI‐34 (1:500, Covance); and mouse monoclonal SMI‐32 (1:500, Covance) that reacts with non‐phosphorylated epitopes of neurofilaments. Species‐specific Alexa Fluor^® 488 and 555 conjugated secondary antibodies (1:500, Life Technologies, Paisley, UK) were used to visualize primary antibody staining. DAPI (4',6‐diamidino‐2‐phenylindole) Vectasheild™ (H‐1200, Vector Laboratories, Peterborough, UK) was used for nuclear identification. Sections were visualized using a Leica (DMI 6000) fluorescence light microscope with a Leica LAS‐AF software and also a Leica TCS SP5‐AOBS confocal laser scanning microscope attached to a Leica DMI 6000 inverted epifluorescence microscope to obtain three‐dimensional images.

MC1 and Alz50 (provided by Peter Davies, Albert Einstein College) are conformation specific mouse monoclonal antibodies that recognize amino acids 7–9 and 313–322 (MC1) [10 (link)] or amino acids 5–15 and 312–322 (Alz50) [11 (link)] of tau and are specific for pathological tau. AT8 (Thermo Scientific, Rockford, IL) is a phosphorylation-dependent mouse monoclonal antibody that recognizes PHF-tau phosphorylated on dual sites Ser202 and Thr205. Other antibodies used in this study that recognize phosphorylated epitopes of tau include AT180 (pThr231; Thermo Scientific), AT270 (pThr181; Innogenetics), and PHF1 (pSer396/pSer404; provided by Peter Davies) [12 (link)]. Antibody TOC1 (tau oligomeric complex 1), which selectively labels tau dimers and oligomers, but does not label filaments [13 (link)] was kindly provided by Lester Binder (Michigan State University). Rabbit polyclonal 17025 is a pan-tau antibody recognizing total mouse and human tau raised against full length recombinant tau [14 (link)]. SMI31 (Covance, Princeton, N.J.) is a mouse monoclonal antibody that reacts with phosphorylated neurofilament H. The anti-actin mouse monoclonal antibody was obtained from the developmental studies hybridoma bank (dshb.biology.uiowa.edu).

The following antibodies were used: APP (Chemicon MAB348), CAII (Said Ghandour); CD3 (Serotec MCA1477);CD3e (Biolegend clone 145-2C11), CD4 (Becton Dickinson clone GK1.5), CD8 (Becton Dickinson clone 53-6.7), CD11b (Biolegend clone M1/70), CD45.2 (Biolegend clone 104), CNP (2′,3′-Cyclic-nucleotide 3′-phosphodiesterase, Sigma C5922), GFAP (Chemicon MAB3402), Iba1 (Wako 019-19741), MAC3 (Pharmigen 01781D); MBP (Serotec MCA409S), Olig2 (Prof Charles Stiles/ Dr. John Alberta, DF308), PCNA (Abcam ab29), SMI31 (Covance SMI-31P), TCF4 (Millipore 04-1080).