Phf 1

Tissue sections were treated as described above. An additional blocking step before the addition of milk was used to reduce endogenous peroxidase activity, using methanol containing 0.3% hydrogen peroxide. Sections were incubated with the respective primary antibody, AT8 (Thermoscientific, 1:600), AT100 (Thermoscientific, 1:500), AT180 (Thermoscientific, 1:500), PHF1 (source: Peter Davies; 1:500) or Aβ-XP (Cell Signaling, 1:200), prior to tracer labelling. Incubation with a secondary biotinylated antibody (either rabbit anti-mouse IgG or swine anti-rabbit IgG; Dako, 1:200, 30 min) was followed by treatment with avidin-biotin complex (Vector Laboratories; 30 min). Tyramine signal amplification (red or green; 1:200) was used as a substrate for horseradish peroxidase. Sections were mounted with either DAPI-containing fluorescent mounting medium or Nissl Neurotrace 640, as described above.

Sections were treated with [¹⁸F]THK-5117 as described above (Phosphorimaging). After tracer incubation, the air-dried sections were dipped in nuclear photographic emulsion K.5 (Ilford; 1:4 dilution in water) and exposed overnight under exclusion of light. Slides were washed in phenisol developer (Ilford; 20%, four minutes), acetic acid (1%, two minutes), hypam fixative solution (Ilford; 20%, four minutes) and water. Immunohistochemistry was subsequently performed as described above. The concentration of the primary antibodies was as follows: AT8 (Thermoscientific; 1:100), Aβ-XP (Cell Signalling; 1:100) and PHF-1 (source: Peter Davies; 1:100). Immunostaining controls not subject to silver dipping were carried out on adjacent tissue sections.
Nuclear emulsion autoradiography photomicrographs were assessed for accumulation of silver clusters over entire areas of brain tissue for each case assessed, including grey and white matter.