Axio imager z1 upright

Mouse back skin and human FUE samples were fixed in formalin, embedded in paraffin and sectioned at 5 µm thickness. In situ hybridization was performed on transverse paraffin sections of the human follicle using ACD RNAscope HD 2.5 duplex assay or 2.5 HD Brown assay with probe HS:VCAN, Hs:AR and HS:PPARGC1a. In situ hybridization on mouse skin was performed with Ms:PPARGC1a and Ms:VCAN probes. Staining was visualized in a traditional light microscope (Zeiss Axio Imager Z1 upright).

Frozen tissue of both mice and human were used for immunofluorescence stainings. Frozen, paraffin fixed mouse tissue sections were melted in room temperature for 5 min, fixed with cold acetone 10 min and washed with PBS. The unfixed, fresh-frozen human tumor biopsies were melted and washed with PBS. The following procedures were same for both tissue types. The unspecific binding of primary antibody was prevented by incubating the sample for 30 min with 0,5% casein in tris buffered saline (TBS) (blocking buffer). Tissue was incubated with primary antibody diluted to blocking buffer at +4 °C overnight. Next, slides were washed with 0,05% Tween in TBS and re-incubated with blocking buffer. Fluorescent labelled secondary antibody and Hoechst for recognizing nuclei were diluted to blocking buffer, administered to the tissue sections and incubated at room temperature for 1 h. Slides were washed with 0,05% Tween in TBS and distilled water. After that slides were mounted with Mowiol mounting medium (Sigma) and covered with cover glass (Menzel). Tissues were analyzed under either Zeiss Axioplan fluorescence microscope, Zeiss LSM 880 Confocal microscope or Zeiss Axio Imager.Z1 upright epifluorescence microscope.