Histofine simple stain

We used three N1-Gal4VP16; UAS-Cre; R26GRR mice (10–13 weeks old), one UAS-Cre; R26GRR mouse (6 weeks old) and one Non-Tg mouse (5 weeks old) for the analysis. To reduce the number of mice used, we used post-weaning single mouse per group for the negative control (UAS-Cre; R26GRR and Non-Tg groups). Prior to sampling the organs, mice were sacrificed by cervical dislocation and perfused with PBS and Mildform 10 N (Cat #: 133-10311, Fujifilm, Osaka, Japan). For fluorescent imaging, 10-µm-thick frozen sections were counterstained with Hoechst 33342 (Cat #: H3570, Invitrogen, Waltham, MA, USA). For immunohistochemistry, 4-µm-thick paraffin sections were sequentially incubated with a rabbit monoclonal anti-Cre recombinase antibody (clone: D7L7L, Cat #: 15036 S, RRID: AB_2798694, Cell Signaling Technology) or a rabbit anti-RFP antibody (cat #: 600-401-379, Lot #: 46317, RRID: AB_2209751, Rockland Immunochemicals, Philadelphia, PA, USA), Histofine SimpleStain (Cat #: 414341, Nichirei Biosciences, Tokyo, Japan), Histofine DAB Substrate Kit (Cat #: 425011, Nichirei Biosciences) and counterstained with Mayer’s hematoxylin solution (Cat #: 131–09665, Fujifilm, Osaka, Japan). Images were captured by using BIOREVO-BZ-X810 (Keyence, Osaka, Japan).

Freshly isolated skin from C57BL/6 mice were immediately fixed in 4% paraformaldehyde in PBS and subsequently embedded in paraffin. Vertical sections, 6 to 8 μm thick, were deparaffinized in xylene and rehydrated with a graded series of ethanol concentrations. Sections were incubated at 4°C overnight with polyclonal anti-TLE3 antibody (Proteintech, Chicago, IL, USA), or normal Rabbit IgG (MBL, Aichi, Japan). After washing, sections were incubated for 1h with peroxidase-labeled secondary antibodies (Histofine Simple Stain)(Nichirei Biosciences, Tokyo, Japan). Diaminobenzidine (Histofine DAB-3S kit)(Nichirei Biosciences) served as the peroxidase substrate. ABZ-9000 (Keyence, Tokyo, Japan) microscope was used for these analyses.