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The immunohistochemistry was performed as reported earlier (Sethi 2013). Briefly tissue sections were de-paraffinized and rehydrated. The tissue peroxidases were inactivated with 0.5 % H₂O₂ in methanol for 20 min. Pepsin (2 mg/ml in 0.01 N HCl) was used to unmask antigen-binding sites (60 min) and then 1 % bovine serum albumin (BSA) in PBS was used to prevent non-specific binding (30 min). Next the tissues were incubated overnight (16 h) at 4 °C with the following antibodies: von Willebrand Factor (1:500, DAKO A0082), Toll-Like Receptor 4 (1:25, IMG-578A, IMGENEX) and Toll-Like Receptor 9 (1:50, IMG-3051, IMGENEX) followed by appropriate secondary antibody (vWF at 1:300 and TLR at 1:100, all from DAKO). VECTOR VIP Peroxidase Substrate Kit (Vector laboratories, Burlingame, CA) was used for colour-development ed followed by counter staining with methyl green (Vector laboratories).

The immunohistochemistry was performed as described earlier [20 ]. Briefly tissue sections were de-paraffinized and rehydrated. The tissue peroxidases were inactivated with 0.5% H₂O₂ in phosphate buffered saline (PBS) for 20 min. Tris-ethylene-diamine-tetraacetic acid buffer was used to unmask antigen-binding sites (30 min) followed by incubation with 1% bovine serum albumin in PBS (30 min) to prevent non-specific binding. Then, the tissues were incubated with the primary antibody for TLR-9 (IMG-3051, IMGENEX; dilution 1:800) followed by appropriate secondary antibody (DAKO catalogue no# R0270 dilution 1:100). VECTOR VIP Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA) was used for color development followed by counterstaining with hemtoxylin (Vector Laboratories).