Telocytes in human skin–are they involved in skin regeneration?

Biopsies of human skin were obtained from three patients (informed written consent). Normal skin samples were obtained from a re-excision procedure after removing a local melanoma. The second excisions were performed according to the Breslow index (tumoural depth), 14 days after primary excision. The samples of normal skin were taken at 1 cm distance from primary suture. Experiments were performed according to the Helsinki guidelines, in full compliance with the Bioethics Committee of the ‘Victor Babeş’ National Institute of Pathology, Bucharest regulations. In situ immunostaining and confocal analysis Paraffin embedded skin samples (7μm thick) were deparaffinised, washed for 30 min. in PBS, pH 7.4 and blocked with 2% BSA. The samples were incubated for 30 min. with 2% normal goat serum (Sigma-Aldrich Chemical, St. Louis, MO, USA). Samples were incubated overnight at 4°C in PBS with either rabbit anti c-Kit or one of the following mouse monoclonal antibodies: anti-vimentin (clone V9, 1:150), anti- CD34 (clone QBEnd-10, 1:25) (both from Dako, Glostrup, Denmark) or anti-nestin (clone 10C2, 1:100) (Millipore, Billerica, MA, USA). In addition, combinations were used in double labelling assays. After washing in PBS with 0.1% (vol/vol) Triton X- 100, the sections were incubated with Alexa Fluor-conjugated, secondary goat anti-rabbit or goat antimouse antibodies (Invitrogen, Molecular Probes, Eugene, OR, USA) for another 2 hrs, at room temperature. Following an extensive washing step, the nuclei were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Negative controls were performed by omitting the primary antibody from the same protocol. Epifluorescence was used to examine three to five immunolabelled sections from each biopsy on a Nikon Eclipse E600 microscope (Nikon Instruments Inc., Tokyo, Japan) with a Nikon Plan Apo 40× objective and the appropriate fluorescence filters. Digital pictures were acquired with a CCD Axiocam HRc Zeiss camera and AxioVision software (Carl Zeiss Imaging solution GmbH, Göttingen, Germany) or with confocal laser scanning microscopy, with a Nikon A1 laser microscope mounted on an ECLIPSE Ti-E inverted microscope. The confocal images were collected with a Plan Fluor 60× oil objective and 1.25-NA water ( z -axis step 0.16 μm). The following lasers and emission filters were used: Ar laser at 488 nm (used for the excitation of Alexa Fluor 488) and emission filter 500-550 nm; 561.2 nm G-HeNe laser (for Alexa Fluor 546) and emission filter 570–620 nm; and 405 nm Laser diode and 425–475 nm emission filter for DAPI. To improve the image quality, some original laser scanning microscopy data were subjected to digital deconvolution and three-dimensional reconstruction with an Imaris × 64 (version 6.3.1.) from Bitplane AG (Zürich, Switzerland). Transmission electron microscopy (TEM) The TEM was performed on small (1 mm 3 ) tissue fragments, processed according to a routine Epon-embedding procedure, as previously described [ 36 ]. Thin sections (about 60 nm) were examined with a Morgagni 286 transmission microscope (FEI Company, Eindhoven, The Netherlands) at 60 kV. Digital electron micrographs were acquired with a MegaView III CCD and iTEM-SIS software (Olympus, Soft Imaging System GmbH, Münster, Germany). To highlight the TCs and Tps, TEM images were digitally coloured in blue with Adobe © Photoshop CS3.