Pannoramic midi digital microscope

The paraffin-embedded sections were deparaffinized and rehydrated. Then, antigens were retrieved in 10 mM boiled citrate buffer (pH 6.0, Beijing Solarbio Science & Technology Co., Ltd., China) for 30 min. Following permeabilization with 0.3% Triton X-100 in PBS for 30 min, the sections were incubated with PBS containing 5% goat serum and 0.3% Triton X-100 for 2 h to block non-specific binding. To evaluate the expression of Rac1, the sections were incubated with mouse anti-RAC1 (1:200, 05-389, Sigma–Aldrich) overnight at 4°C, followed by incubation with FITC-conjugated goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody (1:500, F-2761, Invitrogen, Thermo Fisher Scientific, Inc.) for 2 h at room temperature. After being washed with PBS, the nuclei were counterstained with 4′-DAPI (Sigma–Aldrich) for 5 min. The primary antibody was diluted with blocking buffer, and isotype control antibodies were used as a negative control (Mouse IgG Isotype Control, 10400C, Invitrogen, Thermo Fisher Scientific, Inc.). Images were captured with a Pannoramic MIDI digital microscope (3DHISTECH, Budapest, Hungary) and were analysed using CaseViewer (3DHISTECH, Budapest, Hungary).