Alexa fluor 594 conjugated anti rabbit igg

FaDu cells were seeded onto a MatTek dish (No. 1.5, 35 mm). After the cell culture was at least 70% confluent, the old media was removed. The cell culture was washed gently with PBS and a FL8V1 solution (10 µM) was added. The cells were incubated at 37 °C for 30 min. Subsequently, cells were washed with 2% or 4% KCl solution (10 min at 37 °C) and PBS. Colocalization was performed using Rabbit Early Endosome Antigen 1 antibodies (EEA1) antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-late endosomal/lysosomal marker: Lysosome Associated Membrane Protein 1 (LAMP1) (D2D11) XP rabbit monoclonal antibody (1:500). The Alexa Fluor 594 conjugated anti-rabbit IgG (1:200) (Cell Signaling Technology, Inc., Danvers, MA, USA) was used as a secondary antibody. The cells were imaged with Olympus FluoView 1000 confocal microscope (λEX = 488 and 559 nm for FITC tag and Alexa Fluor 594 excitation, respectively) using the 60× oil immersion objective) and colocalization analysis was performed using Image J (NIH).

3 × 10⁵ OVCAR3 cells were seeded with 3 × 10⁵ CAFs in 6-well ultra-low attachment plates (Corning Cat. No. 3471). At the time of seeding, the plate was kept inclined for 30 min to help the OC cells and CAFs aggregate and interact. Thereafter, plates were reverted to the usual horizontal position and cultured for 7 days to allow the heterotypic spheroids to grow. Spheroid fixation, blocking, and antibody staining were done as described by Condello et al.^{49 (link)}. Briefly, spheroids were fixed and permeabilized in suspension for 3 h at 4 °C in PBS containing 4% PFA (Boston BioProducts BM-155) and 1% Triton X-100 (Thermo Scientific Cat. No. 85112). Spheroids were dehydrated with increasing concentrations of methanol (25%, 50%, 75%, 95%, 100%) and rehydrated in the opposite sequence, then stained with ALDH1 (1:100, BD Bioscience Cat. No. 611194), Wnt5a (1:200, CST Cat. No. 2530) and Vimentin (1:500, Thermo Fisher Scientific Cat. No. PA1-10003) antibodies. Nuclei were visualized by Hoechst 33342 (Life Technologies Cat. No. H3570). The primary antibodies were probed with 1:1000 Alexa Fluor 488 conjugated anti-mouse IgG (Cell Signaling Technology, cat. No. 4408S), Alexa Fluor 594 conjugated anti-rabbit IgG (Cell Signaling Technology, cat. No. 8889) or Alexa Fluor 647 conjugated anti-chicken IgG (Invitrogen, cat. No. A-21449).