Swine anti rabbit igg

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.

To assess treatment effect on the level of Notch2 receptor protein expression, immunohistochemistry was conducted on bone sections collected from the MTX time course study. Briefly, sections were deparaffinized, gradually hydrated and were quenched in 3% H₂O₂ and incubated in Tris EDTA (pH 9) for antigen retrieval. After blocking with 5% Pig serum, 4%BSA, 0.1% Triton-X 100, 0.05% Tween 20 for 60 min, sections were then incubated with primary antibody rabbit anti-Notch2-cleaved N-terminus (Merck Millipore, Darmstadt, Germany) (1:100 in a dilution buffer with 2% BSA and 0.25% Triton-X 100) overnight at 4 °C in a humidified chamber. Reaction was detected with biotinylated secondary antibodies swine anti-rabbit IgG (1:300) (Dako, North Sydney, Australia) for 60 min, and streptavidin-HRP (1:700) (R & D systems, Minneapolis, MN, USA) for 60 min at room temperature. Sections were developed with DAB Plus chromogen (Dako) and counterstained with Hematoxylin [48 (link)]. Replacement of the primary antibody with 1% bovine serum albumin in phosphate-buffered saline (PBS) or normal rabbit IgG at the same concentration was used following the same protocol as a negative control. Images were taken and analysed as previously described [48 (link)].

Cellular proteins were extracted with RIPA buffer (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with protease inhibitor cocktail (Sigma), 100‐mmol·L⁻¹ NaF, and 1 mol·L⁻¹ Na₃VO₄, centrifuged at 15 000 × g for 15 min, and supernatants were collected. Equal amounts of total proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis under reducing conditions using 4–12% gradient gels and transferred onto an Immobilon membrane (Millipore, Bedford, MA, USA). After blocking using 5% nonfat milk in Tris‐buffered saline with 0.1% Tween 20 (TBS‐T), membranes were incubated overnight at 4 °C, with a primary antibody described above. Membranes were washed with TBS‐T and incubated with horseradish peroxidase‐conjugated secondary antibody, goat anti‐mouse IgG (#1706516; Bio‐Rad Laboratories Inc., Hercules, CA, USA, 1 : 5000), or swine anti‐rabbit IgG (P0217; Dako, Glostrup, Denmark, 1 : 5000) diluted in TBS‐T with 1% bovine serum albumin for 1 h at room temperature. The labeled proteins were visualized with a chemiluminescence reagent (PerkinElmer Life Science, Boston, MA, USA).