Coreldraw

Preparations were examined on a Nikon Eclipse Ni microscope equipped with the appropriate filter cubes to distinguish the fluorochromes employed. The images were recorded with a Nikon DS-Qi1Nc digital camera and NIS Elements software BR 4.20.01 (Nikon Instruments Europe BV, Amsterdam, Netherlands). Enteric neuron counts were performed at ×40 magnification. Slight adjustments to contrast and brightness were made using Corel Photo Paint, whereas the figure panels were prepared using Corel Draw (Corel Photo Paint and Corel Draw, Ottawa, ON, Canada).

Statistical analysis was performed using analysis of variance (ANOVA), followed by Tukey HSD tests (p = 0.05) using IBM SPSS Statistics for Windows, version 26.0. Microsoft Office, OriginPro 2020, and Corel Draw were used to calculate and produce the graphs and figures.

Primary data analysis to extract spike times, spike and burst frequencies, and voltage trajectory measures was performed using Spike2 and programs written in its script language. Secondary analyses, statistical tests, and plots were generated in SigmaPlot (version 12.0, Systat Software). Statistical tests used were One Way (1W) or Two Way (2W) Repeated Measures Analysis of Variance (RM-ANOVA), with subsequent Holm–Sidak post hoc comparisons, or Friedman Repeated Measures ANOVA on Ranks (RM-Rank-ANOVA), with subsequent Tukey post hoc comparisons. Significance was assumed at p < 0.05, and is indicated as ^∗ (p < 0.05), ^∗∗ (p < 0.01), and ^∗∗∗ (p < 0.001). Error bars indicate Standard Error of Means. Figures were produced in CorelDRAW (versions X7 and X8, Corel) and Canvas (version 11, ACD Systems).

Cryosections of the heart auricle tissue or cultivated cells were fixed for 20 min using 4% paraformaldehyde (PFA), washed and permeabilized in PBS with TritonX-100 (tissue: 0.2%, cells: 0.02%, Applichem, Darmstadt, Germany) and supplemented with 5% goat serum for 30 min. The applied primary antibodies were diluted in PBS as followed: mouse anti-Nestin 1:200 (Millipore, Burlington, MA, USA), rabbit anti-S100B 1:500 (Dako, Glostrup, Denmark), rabbit anti-α-actinin (Cell-Signaling, Danvers, MA, USA), mouse anti Connexin 43 (Millipore). They were applied for 1 h (cells) or for 2 h (sections), both at room temperature (RT). After three washing steps, secondary fluorochrome-conjugated antibodies (Alexa 555 anti-mouse or Alexa 488 anti-rabbit, Invitrogen, Life Technologies GmbH, Carlsbad, CA, USA) were applied for 1 h at RT with a dilution ratio of 1:300. Nuclear staining was realized by incubation with 4,6-diamidin-2-phenylindol (DAPI) (1 μg/mL, Applichem) in PBS for 15 min at RT. Finally, the samples were mounted with Mowiol (self-made). Imaging was performed using a confocal laser scanning microscope (CLSM 780, Carl Zeiss, Oberkochen, Germany) and image processing was executed with ImageJ and CorelDRAW [25 (link)] (open source and Corel Corporation).

All images were taken using the LSM 700 (Carl Zeiss) confocal microscope, LCI Plan-Neofluar 63×/1.30 glycerol immersion objective at room temperature, and Zen Black acquisition software (Carl Zeiss AG). Image analysis was done using the Zen Blue Lite (Carl Zeiss), PHOTO-PAINT and CorelDraw programs from the CorelDraw Graphics Suite 2017. Postimaging processing was done using Corel PHOTO-PAINT 2017 using the brightness/contrast/intensity adjustment settings equally for images from the same imaging series.

The specimens were examined using a Nikon Eclipse Ni microscope and images were taken using a Nikon DS-Qi1Nc digital camera and NIS Elements software BR 4.20.01 (Nikon Instruments Europe BV, Amsterdam, The Netherlands). Minor adjustments to contrast and brightness were made using Corel Photo Paint, while figure panels were prepared using Corel Draw (Corel Photo Paint and Corel Draw, Ottawa, ON, Canada). The 20× objective was used for morphometric evaluation. In the gastric mucosa, the area occupied by OPs-IR in 4.1 mm² (0.410 × 10 fields) was measured by binarization (described above). In addition, the number of GHR, NPY and SOM IRs in 4.1 mm² were counted in the gastric mucosa.
For each experimental group (CTR, BP 5% and BP 10%), the values obtained for OPs-IR area and the number of EECs were corporate and the means were calculated. The results were expressed as mean ± standard deviation (SD). The data were analyzed by one-way ANOVA (Graph Prism 4, GraphPad Software version 4.01, Inc., La Jolla, CA, USA). The experimental group was considered as the main effect. In addition, the means were then separated using the Tukey-HSD test. A p ≤ 0.05 was considered statistically significant.