Anti 4 hydroxynonenal 4 hne

Liver samples were processed as described.24 In brief, sections of 6 μm were permeabilized with 0.1% Triton X‐100 for 20 minutes and rehydrated with five washes of PBS + 0.5% bovine serum albumin (PBB), and then the tissue sections underwent a blocking step of 45 minutes with PBS + 20% normal goat serum. Samples were incubated with primary antibodies (anti‐4‐hydroxynonenal [4‐HNE] at 1:100, Millipore no. 393207; anti‐lymphocyte antigen 6G [Ly6G] at 1:100, BD Biosciences no. 560599; anti‐F4/80 at 1:250, BD Biosciences no. 552958) in PBB + 0.1 Triton X‐100 for 12 hours or TMR red (1:1,000, Roche no. 12156792910) for 1 hour at room temperature. All immunofluorescent experiments staining sets involved staining β‐actin and nuclear stain for use in quantitation. Imaging conditions were maintained at identical settings within each antibody‐labeling experiment, with original gating performed using the primary depletion control. Large‐area images equivalent to nine unique fields were taken in X and Y with a Nikon A1 confocal microscope (purchased with 1S10OD019973‐01 awarded to Dr. Simon C. Watkins). Quantification was performed using NIS Elements (Nikon, Melville, NY).