Anti mouse f4 80

Tissues were embedded in OCT, frozen on liquid nitrogen, and 7 μm cryostat sections were cut. Specimens were placed on glass slides, air dried and fixed with aceton for 2 min at −20°C. After rehydration with 80% methanol at 4°C, the specimens were washed in PBS and then in PBS with 5% donkey serum and 1% bovine serum albumin, followed by incubation with the respective primary antibodies. Standard H&E and immunofluorescence stainings were performed as described previously [9] (link), [29] (link), using the following antibodies: anti-mouse LYVE-1 (AngioBio), biotin anti-MECA-32 (BD Biosciences), anti-human von Willebrand factor (Dako), anti-mouse podoplanin (clone 8.1.1, Developmental Studies Hybridoma Bank, University of Iowa), anti-mouse keratin 6, anti-loricrin (Covance Research Products), anti-BrdU-Alexa Fluor 594 (Invitrogen), anti-mouse F4/80 (AbD Serotec), anti-mouse CD68 (Abcam), anti-mouse CD8 (BD Biosciences) and anti-mouse CXCR4 (R&D Systems). Alexa Fluor488-, Alexa Fluor594- and Alexa Fluor647-coupled secondary antibodies and Hoechst 33342 were purchased from Invitrogen.

After sacrifice, portions of harvested kidney tissues were fixed in 4% neutral-buffered formalin, followed by dehydration in graded alcohols and embedded in paraffin. For further analysis such as periodic acid-Schiff (PAS) staining or immunostaining 4 μm sections were deparaffinized, rehydrated, transferred into citrate buffer, and either autoclaved or microwave treated for antigen retrieval and processed as described [12 (link)]. For immunostaining following primary antibodies were used: Collagen1α1 (Dako, Hamburg, Germany), anti-mouse CD3, anti-mouse F4/80 (both Serotec, Oxford, UK).

Immunohistochemistry was performed in 1‐μm‐thick paraffin sections of methyl Carnoy's‐fixed specimens using the Vectastain Avidin/Biotin System (Vector Laboratories, Burlingame, CA), as described before 25. Renal tissues were stained using the following primary antibodies: anti‐human Col1A1 (Southern Biotech, Birmingham, AL), anti‐Kim1 (R&D systems, USA), anti‐mouse F4/80 (Serotec, Düsseldorf, Germany), anti‐proliferating cell nuclear antigen (PCNA) (Leinco Technologies, St. Louis, MO) and anti‐Ly6G (BD Biosciences, San Jose, CA). Negative controls for the immunohistochemical procedures consisted of substitution of the primary antibody with non‐immune IgG. Quantification of Ly6G was described before 4. Total number of PCNA‐stained nuclei in renal tubuli was counted in 25 randomly selected fields at ×200 magnification. For staining quantification of collagen and F4/80, 20 consecutive images of renal cortex were taken per section. The percentage of the positively stained area was extracted for intensity using ImageJ software (Wayne Rasband, NIH) and a mean area was calculated. Periodic acid–Schiff (PAS) staining was performed as described before 25, and the tubular injury was scored on a scale of 0–4: 0 = none; 1 = 0–25%; 2 = 25–50%; 3 = 50–75%; 4 = more than 75%. The total score is the calculated average of all tubular scores.

Alanine (ALT) activities (UV test at 37°C) were measured from serum (Roche Modular preanalytics system, Rotkreuz Switzerland). Conventional H&E, Sirius red, and Ladewig staining were performed according to standard protocols [20] (link). Steatosis was scored by an experienced pathologist blinded to experimental data, as previously described [9] (link), [15] (link). Liver sections from fixed paraffin blocks were immunohistochemically stained according to standard procedures using anti-mouse F4/80 and anti-human CD68 (Serotec), respectively [21] (link). CD68 and H&E pictures were analysed by quantifying the area fraction using imaging software in a blinded fashion (ImageJ).

Mouse peripheral blood neutrophils were isolated using a modified version of the negative immunomagnetic separation technique described by Cotter et al.^{44 (link)}. Peripheral blood was incubated with the following monoclonal antibodies: anti-mouse CD2 (1.5 µg/10⁶ lymphocytes), anti-mouse CD5 (2 µg/10⁶ lymphocytes), anti-mouse CD45R (10 µg/10⁶ lymphocytes) (all from Becton Dickinson, Oxford, UK), anti-mouse F4/80 (4 µg/10⁶ monocytes) and anti-mouse CD115 (15 µg/10⁶ monocytes) (both from AbD Serotec, Oxford, UK). Cells were then incubated with goat anti-rat IgG MicroBeads (20 µl/10⁷ cells, Miltenyi Biotec Ltd, Bisley, UK) at 4 °C for 15 min before neutrophils were negatively selected using a magnetic column. Differential counts showed that the neutrophil purity was between 90–98%.
For human studies, all experiments were approved by University of Sheffield Research Ethics Committee (reference SMBRER310) and were performed in accordance with the relevant guidelines and regulations. All subjects gave informed consent. Human peripheral blood neutrophils were isolated using density gradient separation as previously described^{45 (link)}.