Anticholine acetyltransferase

After the in vivo experiments were completed, all animals were humanely euthanized, and their hearts were removed for histologic examination. Tissue samples from Koch's triangle were excised according to the anatomic and ablation markers, fixed with 4% paraformaldehyde, embedded in paraffin wax, and cut into 10‐μm‐‒thick cross‐sections. Slides were stained for microscopic examination with hematoxylin and eosin, anticholine acetyltransferase (Abcam, Cambridge, MA), and antityrosine hydroxylase (EMD Millipore, Darmstadt, Germany). The number of visible nuclei was factored for the square area of the GP visible in each field with ImageJ software (National Institutes of Health, Bethesda, MD).

Reagents were purchased from Sigma (Italy). Antibodies raised in rabbit: anti‐arginase 1 (ARG1) (1:100, Abcam, UK); anti‐CD163 (1:100, Santa Cruz Biotechnology, USA); anti‐choline acetyltransferase (1:500, AbCam); anti‐DAO1 (1:200, Bioss, USA); anti‐H1R (1:200, Alomone, Israel); anti‐H2R (1:200, MyBioSource, USA); anti‐H3R (1:200, Alomone); anti‐H4R (1:200, Santa Cruz Biotechnology); anti‐HDC (1:20, AbCam); anti‐HNMT (1:200, Sigma); anti‐inducible nitric oxide synthase (iNOS) (1:1000, CST, USA); anti‐myelin basic protein (MBP) (1:1000 CST); anti‐nuclear factor‐kappa B (NF‐κB) p65 (1:500, CST); anti‐phospho‐AKT (1:500, CST); anti‐phospho‐p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, CST); and anti‐phospho‐NF‐κB p65 (Ser536) (1:500, CST). Antibodies raised in mouse: anti‐AKT (1:500, Santa Cruz Biotechnology); anti‐GAPDH (1:2500, Calbiochem, USA); anti‐gp91^phox (1:1000, BD Transduction Laboratories, USA); anti‐SMI32 (1:1000, Covance, USA); and anti‐p44/42 MAPK (ERK1/2) (L34F12) (1:1000, CST). Antibodies raised in rat: anti‐CD11b (1:200, AbD Serotec, UK).