Methods and Reagents

Eight-micron thick paraffin-embedded sections are stained with:
- hematoxylin-eosin (H&E)
- Bielschowsky silver stain
- Sevier-Munger silver stain
- thioflavine S
- cresyl violet
- myelin stains(e.g. LFB-PAS)
Additional paraffin-embedded sections are immunohistochemically labeled with antibodies to:
- alpha-synuclein
- β-amyloid (Aβ)
- FUS (fused in sarcoma)
- glial fibrillary acidic protein (GFAP)
- neurofilaments
- tau (microtubule associated protein)
- Tar DNA-binding Protein 43 (TDP-43)
- phospho-Tar DNA-binding Protein 43 (TDP-43)
- tyrosine hydroxylase
- ubiquitin
Endogenous perox idase in tissue sections is blocked with 3% H2O2 in methanol for 5 min at 40°C, and ______ sections are pretreated _______. For immunodetection, nonspecific reagent binding is blocked with normal serum in 0.1 M Tris each for one hour at room temperature. Sections are incubated in primary antibody (_______) _______. After rinsing, sections are incubated for _______ in biotinylated secondary antibody, rinsed, incubated for _______ in avidin-biotin complex, and then developed with diaminobenzidine (DAB). Appropriate positive controls are included in each immunohistochemical experiment. Negative controls consist of sections incubated without primary antibody.
- Phosphorylated alpha-synuclein mouse monoclonal antibody Clone pSyn #64 – Wako, Richmond, VA
- Aβ mouse monoclonal antibodies 6E10 and 4G8 – Covance, Princeton, NJ
- FUS rabbit polyclonal (Prestige) antibody – Sigma-Aldrich, St. Louis, MO
- Glial fibrillary acidic protein, mouse monoclonal and rabbit polyclonal antibodies – Dako, Carpinteria, CA
- Neurofilaments, phosphorylated, mouse monoclonal antibody – Dako, Carpinteria, CA
- Tau antibody – mouse monoclonal PHF-1 – gift from Peter Davies
- TDP-43 rabbit polyclonal antibody – Protein Tech, Chicago, IL
- Phospho-TDP-43 rabbit polyclonal antibody – Cosmo, Carlsbad, CA
- Tyrosine hydroxylase mouse monoclonal antibody – Sigma-Aldrich, St. Louis, MO
- Ubiquitin mouse monoclonal antibody Clone Ubi-1 – Millipore, Billerica, MA
- Pepsin, ready-to-use – Accurate Chemical & Scientific, Westbury, NY
- Vectastain Elite kits (Vector Laboratories, Burlingame, CA) are used for ABC-based immunodetection of antigen-antibody complexes.
- Diaminobenzidine - (Vector Laboratories, Burlingame, CA) is used for color development