Alexa fluor 594 conjugated anti mouse igg

For triple fluorescence analysis, cells were fixed with 4% paraformaldehyde and then permeabilized by 0.5% (v/v) Triton X-100 as reported [32 (link)]. The following primary and secondary antibodies were used: mouse MAb anti-HPV16 E7 antibody (Cervimax), rabbit PAb anti-gelsolin (Novus Biologicals), AlexaFluor 488-conjugated anti-rabbit (Invitrogen), AlexaFluor 488-conjugated anti-mouse IgG (Invitrogen Corporation), and AlexaFluor 594-conjugated anti-mouse IgG (Invitrogen). For F-actin detection, cells were stained with TRITC-phalloidin (Sigma, St Louis MO, USA) for 30 min at room temperature. After washing, all samples were counterstained with Hoechst 33258 (Sigma, 1 mg/ml in PBS) and then mounted in glycerol/PBS (ratio 1:1, pH 7.4). The images were acquired by intensified video microscopy (IVM) with an Olympus fluorescence microscope (Olympus Corporation of the Americas, Center Valley, PA.), equipped with a Zeiss charge-coupled device (CCD) camera (Carl Zeiss, Oberkochen, Germany).

HFL-1 cells (2.0 × 10⁴ cells) were cultured in 24-well plates (DMEM supplemented with 10 mM HEPES). TGF-β1 (10 ng ml⁻¹) (no. 100-21C, Peprotech) was added. Next, the cells were fixed with 4% PFA for 15 min on ice and then permeabilized and blocked in PBS containing 0.3% Triton X-100 and 3% BSA for 1 h at room temperature. The fixed cells were incubated overnight with anti-collagen type I (SP1. D8) antibody diluted with PBS containing 0.3% Triton X-100 and 1% BSA (1 : 100) at 4°C. After washing with PBS, the cells were stained with Alexa Fluor 594-conjugated anti-mouse IgG (diluted 1 : 400, no. A11005, Invitrogen). The cells were washed with PBS and incubated overnight with 10 µM Py₃-FITC in PBS at 4°C. After washing with PBS three times, the cells were mounted with PBS containing 50% glycerol.

Immunohistochemistry and immunofluorescence analysis were performed as previously described^{22 (link)}. The paraffin-embedded endometrial tissues were blocked with 10% normal serum in PBS (pH 7.5) and then incubated with antibodies against SIRT1 (9475 for IHC and 8469 for IF, Cell Signaling), BCL6 (14895, Cell Signaling), KRAS (ab55391, Abcam) and GLI1 (sc20687; Santa Cruz Biotechnology). For immunohistochemistry, sections were incubated with secondary antibody conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA). Immunoreactivity was detected using the Vectastain Elite DAB kit (DAB-Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. A semi-quantitative grading system (H-score) was used to compare the immunohistochemical staining intensities as previously described^{83 (link)}. For immunofluorescence, the sections were exposed to primary antibodies overnight at 4 °C and secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG (Invitrogen, Grand Island, NY) and Alexa Fluor 594-conjugated anti-mouse IgG (Invitrogen) for 2 hour at room temperature. 4′,6‐diamidino‐2‐phenylindole (DAPI; Vector Laboratories) was used to enable nuclear visualization. The IgG antibody was intended for use as a negative control with SIRT1 and BCL6 proteins in the women endometrium (Supplementary Fig. S3).

For mitochondria labeling, living cells were incubated with 5 µM MitoTracker red (Invitrogen) for 45 min at 37 °C. After this time, the cells were washed in PBS, fixed in 4% paraformaldehyde and counterstained with Hoechst 33258. For YAP, TBC1D2, LC3 and p62 staining, the cells were fixed with 4% paraformaldehyde, permeabilized by 0.5% (v/v) Triton X-100 and incubated for 1 h at 4 °C with specific primary antibodies: LC3 (Novus Biologicals; 1:100); p62 (Cell Signaling Technology; 1:100); YAP (Santa Cruz Biotechnology; 1:100); and TBC1D2 (Thermo Fisher Scientific; 1:100). AlexaFluor 488-conjugated anti-mouse IgG and AlexaFluor 594-conjugated anti-mouse IgG (both Invitrogen) were used as secondary antibodies. After washing, all samples were counterstained with Hoechst 33258 and then mounted in fluorescence mounting medium (Dako, Glostrup, Denmark). Images were acquired with intensified video microscopy (IVM) with an Olympus fluorescence microscope (Olympus Corporation, Milan, Italy) equipped with CoolLed pE-300-W (CoolLED Ltd., Andover, UK).

Frozen sections of teratomas were prepared for immunostaining as described previously [32 (link)]. We used Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 594-conjugated anti-mouse IgG (Invitrogen) as secondary antibodies. Nuclei were counterstained with DAPI.

Cells were fixed with 4% paraformaldehyde, permeabilized by 0.5% (v/v) Triton X-100 [24 (link)] and incubated for 1 h at 4 °C with primary antibodies. The following primary and secondary antibodies were used: anti-AMOT1, anti-YAP, anti-p-YAP, AlexaFluor 488-conjugated anti-rabbit (Invitrogen, Carlsbad, CA, USA; #A11034), AlexaFluor 488-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA, USA; #A11001) and AlexaFluor 594-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA, USA; #A11005). For F-actin detection, cells were stained with TRITC-Phalloidin (Sigma-Aldrich, St Louis, MO, USA; #P1951) for 30 min at room temperature. After washing, all samples were counterstained with Hoechst 33258 (Sigma-Aldrich, St Louis, MO, USA; #861405) and then mounted in fluorescence mounting medium (Dako, Glostrup, Denmark; #S3023). Images were acquired by intensified video microscopy (IVM) with an Olympus fluorescence microscope (Olympus Corporation of the Americas, Center Valley, PA, USA), with a Zeiss charge-coupled device camera (Carl Zeiss, Oberkochen, Germany).