X3p 1gal

Paraffin embedded tissues were cut into 4 µM sections and placed on glass slides. The slides were heat fixed at 60 °C for at least thirty minutes, then dried at 60 °C in an oven for 20–60 min. The sections were deparaffinized with xylene (Fisher Scientific, X3P-1Gal) and rehydrated through ethanol series (Fisher Scientific, 04-355-223). Slides were submerged in hematoxylin (Sakura, 6190) for four minutes, then rinsed with running tap water for two minutes. Slides were submerged in acid alcohol (Sakura, 6190) for 10 s, then rinsed with running tap water for two minutes. Slides were submerged in bluing reagent (Sakura, 6190) for 10–30 s, followed by a rinse with running tap water for two minutes. Slides were submerged in 95% ethanol for 30 s and then Eosin-Y (Sakura, 6190) for two minutes, followed by 95% ethanol for 30 s. Slides were then submerged in 100% ethanol for one minute, twice. Slides were submerged in xylene for 2 min, twice. A coverslip was then placed on slides using Permount (Fisher Scientific, SP15-500).

Heart samples were fixed in 4% paraformaldehyde (sc‐281,692, Santa Cruz) and were embedded in paraffin. Sections were cut (8 μm) and re‐hydrated through sequential incubations in a series of graded xylene (X3P‐1GAL, Fisher Scientific), ethanol (BP2818, Fisher Scientific), and phosphate buffer solution (PBS). Antigen retrieval was performed by running slides submerged in IHC‐TekTM Epitope Retrieval Solution. Slides were blocked with 15% horse serum (S‐2000, Vector Laboratories) for 1 h and followed by incubation with anti‐MPO antibody (1:400, ab208670, Abcam). DAB labeling was performed with HRP‐conjugated donkey anti‐rabbit IgG (1:250, A10040, Invitrogen). Hematoxylin (H‐3404, Vector Laboratories) was used for nuclear staining. For the result quantification, 10 pictures were taken from each sample and the average number of cells per picture was calculated.