For p65 immunodetection, a slice of the lung of each mouse was deparaffinized in Histo-clear and antigen retrieved by incubating the slides in 1 mM EDTA pH 8.0 at 98 °C for 20 min.
Negative sections were prepared by incubating the slides in the absence of the primary antibody.
The color was developed by incubating the slides for 20 min with 3,3-diaminobenzidine (Zymed Laboratories).
Endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide for 15 minutes at room temperature.
Antigen retrieval was performed by incubating the slides in 100% formic acid for 5 minutes, followed by extensive washes.
Prior to hybridization of single-stranded template DNA to the microarrays, unreacted epoxy groups were blocked by incubating the slides for 20 minutes at 50°C in 0.1 M Tris-HCl pH 9.0 containing 50 mM ethanolamine and 0.1% SDS.
Immediately before use, the printed PAMPs microarrays were rinsed with PBS-T and then blocked by incubating the slides in 3% BSA in PBS at 37°C for 1 h.
After fuctionalization the slides were rinsed in DMF, the Fmoc protecting group removed by incubating the slides in piperidine (10% v/v) in DMF for 30 min, and the slides were subsequently rinsed in DMF.
Epitope demasking was carried out by incubating the slides in buffer citrate 10 mM, PH 6 (0.1 M sodium citrate, 0.1 M citric acid) in EZ- RETRIEVER (Biogenex, San Ramon USA).
Endogenous peroxidase activity was quenched by incubating the slides in peroxidase blocking reagent for 10 minutes.
Spirochetes were fixed by incubating the slides in cold acetone for 10 minutes at -20ºC.
