Western blotting and immunogold EM were carried out as described19 (link). For Western blotting, samples were resolved on 4–20% or 10% Tris-glycine gels (Novex), and the primary antibodies were diluted in PBS plus 0.1% Tween 20 and 1% BSA. BR133, BR134 and BR13510 (link) at 1:4000; AT8 (Thermo; catalogue nr. MN1020) at 1:1000; MC-128 (link) at 1:10; HT7 (Thermo; catalogue nr. MN1000) at 1:2500, Anti 4R-tau (Cosmo Bio; catalogue nr. CAC-TIP-4RT-P01) and TauC425 (link) at 1:2000; and MN42324 (link) at 1:500. For immunogold EM, primary antibodies were diluted as follows: BR133, BR134, BR135, AT8, Anti 4R, TauC4 and MN423 at 1:50; and MC-1 at 1:10. Neurohistology, immunohistochemistry, and molecular genetics were carried out as described49 (link). For immunohistochemistry, the following antibody dilutions were used: RD3 (Millipore; catalogue nr. 05-803) at 1:3000; Anti-4R at 1:100; and AT8 and AT100 (Thermo; catalogue nr. MN1060) at 1:300. The brain sections were 8 μm thick, and were counterstained with haematoxylin.
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Tau Protein Detection Protocol
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Other organizations : MRC Laboratory of Molecular Biology, Indiana University – Purdue University Indianapolis
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Variable analysis
independent variables
- Primary antibody dilutions for Western blotting
- Primary antibody dilutions for immunogold EM
- Tau protein detection and localization
- Resolution of samples on 4–20% or 10% Tris-glycine gels (Novex) for Western blotting
- Dilution of primary antibodies in PBS plus 0.1% Tween 20 and 1% BSA for Western blotting
- Brain section thickness of 8 μm for immunohistochemistry
- Haematoxylin counterstaining of brain sections for immunohistochemistry
- BR133, BR134, BR135 antibodies at 1:4000 dilution
- AT8 antibody at 1:1000 dilution
- MC-1 antibody at 1:10 dilution
- HT7 antibody at 1:2500 dilution
- Anti 4R-tau antibody at 1:2000 dilution
- TauC4 antibody at 1:2000 dilution
- MN423 antibody at 1:500 dilution
- Not explicitly mentioned
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