Goat anti s100a9

Left lobes of liver tissues and small intestines were excised and fixed in 4% paraformaldehyde (Santa Cruz Biotechnology) overnight at 4°C. Ten-micron paraffin-embedded sections were prepared and slides were boiled in Diva Decloaker solution (Biocare Medical; #DV2004) in a pressurized chamber for 15 min. Sections were blocked with in PBS containing 5% donkey serum, 1% bovine serum albumin (BSA) (Sigma-Aldrich) and 0.03% Triton-X (Plusone, #17-1315-01) for 1 hour, then incubated in with rat anti-F4/80 (Abcam, #ab6640), goat anti-S100A9 (R&D Systems, #AF2065), rabbit anti-von Willebrand Factor (DAKO, #a0082), rat anti-PV1 (BD pharmingen, #550563), or rabbit anti-ABCA1 (Novus Biologicals, NB400-105) at 4°C overnight. Primary antibodies were detected using Cy3- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch). The stained sections were imaged using an SP8 confocal microscope (Leica) equipped with nine lasers and four tunable detectors (two hybrid, two tunable) and a 20X HC PL Apo CS 2 multi-immersion objective, NA 0.75. Images were processed with Imaris software (Bitplane). Ten fields were quantified and averaged for each sample, with cell counts per image quantified using Image J software (NIH). All slides were analyzed by in blinded and randomized fashion.