Alexa fluor 594 conjugated goat anti mouse

Anti-Rhodopsin (MS-1233-P; Ab-1 (RET-P1); Neomarkers, Fremont, CA, USA); anti-trkA^NGFR (sc-118; Santa Cruz Biotech, Santa Cruz, CA, USA); anti-phospho trkA^NGFR (p-trkA^NGFR; sc-130222; Santa Cruz); anti-p75^NTR (sc-6188; Santa Cruz); and anti-GAPDH (sc-365062; Santa Cruz). Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-mouse as well as horseradish peroxidase-conjugated anti-rabbit/mouse specie-specific secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).

De-identified HGSOC patient tumors collected during surgery by the Indiana University Simon Cancer Center’s Tissue Procurement & Distribution Core were obtained from the core using an institutionally approved ‘non-human subjects research protocol’ (Protocol # 1606070934). Freshly collected tumors were embedded in OCT compound (Tissue-Tek Cat. No. 4583), flash-frozen, and stored at −80 °C. 12 µm tumor sections were made using a cryo-microtome (Leica CM1850), fixed with 4% PFA for 15 min at 37 °C, permeabilized with 1X Proteinase K solution (provided in the Click-It TUNEL Assay kit) followed by TUNEL staining using Click-iT TUNEL Alexa Fluor Imaging Assay for Microscopy (Thermo Fisher Scientific Cat. No. C10245). The OC cells were labeled with Pan-Keratin (1:200, Cell Signaling Technology, Cat. No. 4545S) and CAFs with αSMA (1:200, Cell Signaling Technology, Cat. No. 19245 S). Alexa Fluor 594 conjugated goat-anti-mouse (1:1000, Cell Signaling Technology, Cat. No. 8890S) and Alexa Fluor 647 conjugated goat-anti-rabbit (1:1000, Cell Signaling Technology, Cat. No. 4414S) were used to detect the primary antibodies and nuclei were labeled with Hoechst 33342 (1:10,000, Life Technologies Cat. No. H3570). The slides were mounted with ProLong Gold (Invitrogen Cat. No. P36930), and the images were acquired with a Leica SP8 confocal microscope.