Application suite version 2

Tissue specimens were obtained through a skin biopsy, using the “Punch” technique performed by a gynecologist. The authors performed a sample withdrawal in each group 30 days after the application of the second therapy session and histological and immunohistochemical analysis was performed.
Specimens of control healthy and treated skin samples of the adjacent area were obtained using 2-mm biopsy punches 60 days after treatment. Each sample was immediately placed in 10% neutral buffered formalin and processed for histotechnical analyses as described previously. Sections (5 μm) were cut, placed onto slides, stained with hematoxylin and eosin, and with Picrosirius Red (for 1 h). An attached camera showed the stained tissues under a binocular light microscope (Olympus CX31, Hamburg, Germany). Photomicrographs of various microscopic fields were taken at different magnification levels (× 40, × 100, or × 400), and the collagen content was measured with a Software Leica Application Suite, version 2.8.1 (Leica Microsystems GmbH, Wetzlar, Germany).

Paraffin-embedded tissue blocks were cut at a thickness of 7 µm and slices were mounted on glass slides, deparaffinized with xylene and rehydrated in a descending alcohol series (100%, 96% and 70%). Slices were boiled in citrate buffer for antigen unmasking, were blocked for 60 min at room temperature with 1% BSA in PBS and were incubated overnight at 4 °C with rabbit anti-CHGA or anti-SYP antibody (1:200 in PBS with 0.5% BSA). After three washing steps with PBST, slices were incubated for 1 h at room temperature in PBS/0.5% BSA with diluted (1:200) secondary biotinylated swine anti-rabbit IgG. After washing three times with PBST, signal enhancement was achieved by Vectastain^® (Linaris, Wertheim-Bettingen, Germany) according to the manufacturer instructions. Immunoreaction was visualized with diaminobenzidine (DAB) (Sigma), followed by nuclear staining using hematoxylin solution (Thermo Fisher Scientific, Oberhausen, Germany). Finally, slices were dehydrated and mounted with Xylene Substitute Mountant (Thermo Fisher Scientific, Oberhausen, Germany). Slides were scanned using a Leica DM4000 B microscope, Leica DFC290 camera and analyzed using the Leica Application Suite version 2.8.1 (Leica Microsystems, Wetzlar, Germany).