4.6. F4/80 Staining

Macrophage staining of livers were assessed in slides of frozen sections (5–10 µm). Slides were fixed in ice cold ethanol for 15–20 min. Slides were air-dried for 10 min and washed in PBS 2 times for 2 min. Thereafter, slides were quenched in 0.3% H₂O₂ for 10 min. PBS washing was repeated for 2 min and air dried. Then, 1–2 drops of Avidin solution (Abcam, Cambridge, UK) were added on each slide and incubated for 10 min. The washing procedure was repeated for 2 min. Next, 1–2 drops of Biotin blocking solution (Abcam) were added to each slide and incubated for 10 min at room temperature. After 2 times of washing in PBS, slides were incubated with 10% FCS for 30 min. Afterwards the diluted anti-F4/80 antibody (Merck, Taufkirchen, Germany) was given to slides and incubated for 2 hours. The slides were washed twice in PBS for 4 min and air dried. The secondary antibody was added to the slides and incubated for 1 hour. After washing the slides three times in PBS and air drying, the ABC reagent was added for 30 min before washing the slides three times with PBS. DAB solution (Vector Laboratories, Burlingame, CA, USA) was added to the slides and incubated for 5 min. Slides were washed and stained in hematoxylin for 5 min and washed two times for 2 min. Slides were air-dried before mounting.