2.3. Immunohistochemical Analysis

Serial cryostat sections (with a thickness of 10 μm, Frigocut, Reichert-Jung, Nussloch, Germany) of the CaMG were placed on chrome alum-coated slides. The presence of FB-positive perikarya was checked in serial sections of the bilateral CaMGs using an Olympus BX51 microscope (Olympus, Warsaw, Poland), equipped for epi-illumination fluorescence microscopy (V1 module: excitation filter 330−385 nm, barrier filter 420 nm). Sections with FB-positive perikarya were used for double-labeling immunofluorescence to determine DβH and/or NPY, SOM, GAL and VIP immunoreactivity [34]. The sections were dried (at 32 °C, 45 min), rinsed in a phosphate buffer with 0.8% sodium chloride and 0.02% potassium chloride (PBS, 3 × 10 min) and incubated in 10% normal goat serum in PBS with 0.3% Triton X-100 (Sigma, Saint Louis, MO, USA) and 1% bovine serum albumin (BSA; Sigma, USA) for 20 min. Next, the sections were incubated overnight (at 4 °C) with primary antibodies diluted in PBS containing 0.3% Triton X-100 and 1% BSA, raised against DβH (rabbit polyclonal, Cat. # AB1585, Merck Millipore, Kenilworth, NJ, USA, 1:500) and/or NPY (mouse monoclonal, Cat. # ABS 028-08-02, ThermoFisher Scientific, Waltham, MA, USA, 1:1000), SOM (rat monoclonal, Cat # 8330-0009, AbD Serotec, Kidlington, UK, 1:60), GAL (guinea pig polyclonal, Cat. # T-50-36, Penisula, San Carlos, CA, USA, 1:800) or VIP (mouse polyclonal, Cat # 9535-0504, BioGene Ltd., Huntingdon, UK, 1:2000). On the next day, the sections were rinsed (PBS, 5 × 15 min) and incubated with secondary antibodies (in PBS with 0.25% BSA and 0.1% Triton X-100) for 4 h (Alexa Fluor 488 nm donkey anti-rabbit, Cat # A21206, Alexa Fluor 546 nm donkey anti-mouse, Cat # A10036, Alexa Fluor 546 nm donkey anti-rat, Cat # A11081 and Alexa Fluor 546 nm donkey anti-guinea pig, Cat # A11074, all from ThermoFisher Scientific, Waltham, MA, USA, and diluted 1:1000) to visualize the following antibody combinations: DβH/NPY, DβH/SOM, DβH/GAL and DβH/VIP. After that, the sections were rinsed (PBS, 3 × 5 min) and covered with a polyethylene glycol/glycerin solution with 1,4-diazabicyclo[2.2.2]octane (DABCO, Sigma, USA). To control for immunofluorescence specificity, standard tests (pre-absorption for the applied antisera with the respective antigen at a content of 20–50 μg antigen/mL diluted antiserum, exclusion of primary or secondary antisera and replacement by non-immune sera of all the primary antisera used) were conducted. The specificity of retrograde tracing was examined by the use of the various tests presented previously [32]. FB-positive and double-immunostained perikarya were investigated and photographed with the appropriate filter sets for fluorescein isothiocyanate (FITC, B1 module, excitation filter 450–480 nm, barrier filter 515 nm) and CY3 (G1 module excitation filter 510–550 nm, barrier filter 590 nm). DβH-, NPY-, SOM-, GAL-, VIP-immunoreactive and/or all FB-positive perikarya were calculated in every fourth section of the bilateral CaMGs. CaMG uterine perikarya profiles with a visible nucleus were only scored to avoid double counting. The perikarya distribution was determined for particular regions of CaMG, taking into account the method reported previously [34]. In brief, the cranial region of the CaMG constitutes an area corresponding to I splanchnic lumbar nerve and intermesenteric nerve, the dorsal region forms the area corresponding to II and III splanchnic nerves, the caudal region composes the area corresponding to IV splanchnic lumbar nerve and hypogastric nerve and the ventral region constitutes the area corresponding to caudal colonic nerves. In addition, the perikarya were classified as small (with a diameter of 23 ± 10 μm) or large (with a diameter of 51 ± 17 μm) [34], using Cell F Imaging Software (Olympus, PL). The perikarya sizes were estimated by measurements of their long and short axis. The average value of the perikaryon diameter was assigned by the use of the equation d = √l × k, where d equals the diameter of a circle with a surface area which is the most similar to the surface area of an ellipsoidal figure with a long axis (l) and a short axis (k) [35]. By adding the small and large perikaryal cell count from all areas of the CaMG, the total population of perikarya for each studied group was determined. The images were captured by a digital camera connected to a PC and analyzed with AnalySIS software (version 3.02, Soft Imaging System, Münster, Germany).