Metal enhancer

Mice whole brain samples were coronally cut into 10 μm sections with a paraffin microtome, and mounted on glass slides pretreated with (3-Aminopropyl)-triethoxysilane.
Slides were deparaffinized by immersion in xylene (2x 15 min), and rehydrated using graded ethanol (2x 100% for 5 min; 95% for 5 min; 90% for 5 min; 80% for 5 min; 70% for 5 min), followed by washes with distilled water (2x 5 min) and phosphate-buffered saline (PBS, 2x 5 min). For antigen retrieval slides were boiled for 15 min in 20 mM citrate buffer (pH 6). After three washes with tris-buffered-saline (TBS), slides were incubated overnight at room temperature (RT) with primary antibody for TH diluted 1:500 in TBS. Slides were washed three times in TBS, and then incubated 2 h at RT with the specific secondary-HRP antibody diluted 1:200 in TBS. After washing three times with TBS, Signal was developed using SIGMAFAST DAB (3,3′-Diaminobenzidine tetrahydrochloride) with metal enhancer (Sigma Aldrich). After washing in distilled water, slides were dehydrated in graded ethanol dilutions, and xylene and permanently mounted with Eukitt (O. Kindler, Freiburg, Germany).

pCDFDuet1 containing traB/virB4_C-His and traG/virB11_C-Strep was used as a template to design primers and introduce point mutations (Q8D, R54E, N55E and K58E) in TraG and (E591R, E594R, A598E and Y619R) in TraB using PCR and In-Fusion cloning (Takara Bio). After the lysate-clarification step described above, supernatants were loaded onto a GE Healthcare HisTrap HP 5 ml column pre-equilibrated with buffer D (50 mM Tris pH 8, 150 mM NaCl, 1 mM DTT). After extensive wash with buffer D followed by a wash with buffer D supplemented with 20 mM imidazole, the recombinant protein was eluted in a gradient of high imidazole buffer (50 mM Tris pH 8, 150 mM NaCl, 1 mM DTT and 300 mM imidazole) and analysed on SDS–PAGE, adjusting load volumes so as to have equal amounts TraB on the gel. Western blot was performed to confirm the identities of TraB and TraG using a Bio-Rad mini-blot module and the two proteins forming the complex were probed using horseradish peroxidase (HRP) conjugated anti-His (Sigma Aldrich; 1:2,000 dilution)) and anti-Strep (EMD Merck; 1:4,000 dilution) antibodies and visualized by incubation with SigmaFast DAB with metal enhancer (Sigma Aldrich). Expression of TraG wild-type and mutants was assessed by comparing the corresponding TraG band in induced and non-induced cells. Non-edited pictures of gels and westerns are shown in the supplementary information.