Donkey anti rabbit igg fitc

Western blot analysis was performed as previously described [19 (link)]. Primary antibodies against occludin (c-term), ZO-1, and claudin-5 and secondary antibodies, Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 555 donkey anti-rabbit, were purchased from Thermo Fisher Scientific. To detect the formation of AGE products with MG-modification, mouse anti-methylglyoxal monoclonal antibodies (STA-011; Cell Biolabs, San Diego, CA, USA) were used. Primary antibodies against p-mTOR, t-Akt, p-Akt, t-S6K1, p-S6K1, HIF-1α, COXIV, VDAC, and Parkin (Prk8) were purchased from Cell Signaling Technology (4695S, Danvers, MA, USA). Primary antibodies against t-mTOR and occludin (E-5) and the secondary antibody donkey anti-rabbit IgG-FITC were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The primary antibody against LC3B was purchased from Sigma Aldrich. Primary antibodies against Glo-1 and Glo-2 were purchased from Abcam. To counterstain the total protein in the gel, Coomassie Brilliant Blue staining was used as previously described [8 (link)].

Metastasis-derived CAFs were seeded in Lab-Tek II chamber slides at a density of 1 × 10⁵ per well and incubated for 24 h in the corresponding maintenance media. For immunofluorescence staining, cells were transfected for 24 h, fixed in 4% paraformaldehyde, permeabilized with 0.1% TWEEN three times for 5min and then were blocked for 30 min at room temperature with PBS containing 10% normal donkey serum (Santa Cruz Biotechnology, DBA, Milan, Italy), 0.1% Triton X-100, and 0.05% TWEEN. Thereafter, cells were incubated overnight at 4 °C with a primary antibody against GPER (K-19) (1:100 purchased from Santa Cruz Santa Cruz Biotechnology, DBA, Milan, Italy) in PBS containing 0.05% TWEEN. After incubation, the slides were extensively washed with PBS and incubated with donkey anti-rabbit IgG-FITC (1:100, from Santa Cruz Biotechnology, DBA, Milan, Italy) and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (1:1000, Sigma-Aldrich, Milan, Italy). The slides were imaged on the Cytation 3 Cell Imaging Multimode reader (BioTek, Winooski, VT) and analysed using the software Gen5 (BioTek, Winooski, VT).