Everbrite hardset mounting medium with dapi

GH3, A7, and M2 cells were seeded on 13-mm poly-L-lysine-coated coverslips at a density of 1.25 × 10⁵ cells/well in 24-well plates and grown at 37°C for 18 h. The following day, cells were incubated with pasireotide 100 nM or octreotide 100 nM for 0, 5, or 30 min. For immunofluorescence analysis of SST₂ and SST₅ localization in GH3, A7, and M2 cells, rabbit anti-SST₂ UMB1 #ab134152 (1:50, Abcam, Cambridge, UK) and mouse anti-SST₅ #6675-1-Ig (1:200, Proteintech, Rosemont, IL, USA) antibodies were used and incubated o/n at 4°C. Anti-mouse Alexa Fluor™ 546-conjugated secondary antibody (1:500, Thermo Fisher Scientific, CA, USA) and anti-rabbit Alexa Fluor™ 488-conjugated secondary antibody (1:500, Thermo Fisher Scientific, CA, USA) were incubated at room temperature for 2 h. All antibodies were diluted in Antibody Diluent Reagent Solution (Life Technologies, Thermo Fisher, CA, USA). Coverslips were mounted on glass slides with EverBrite™ Hardset Mounting Medium with DAPI (Biotium, Fremont, CA, USA) for subsequent observation under epifluorescence microscope. The NIH ImageJ software was used to merge single-channel images before and after deconvolution.

HCT116 cells exponentially growing on sterilized cover glass slides (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) were treated with gefitinib, with or without NDAT or PI3K inhibitor (LY294002) (Selleck Chemicals). The cells were immediately fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min. Fluorescein-labeled Sambucus nigra lectin (FITC-SNA, Vector Laboratories, Burlingame, CA, USA) that preferentially binds to α2,6-linked sialic acid structure was used to investigate the product of ST6Gal1 after these treatments. Cells on the slides were incubated with anti-EGFR antibody (GeneTex) overnight at 4 °C and then incubated with Alexa Fluor®-647-conjugated secondary antibody (Abcam, Cambridge, UK) for 1 h at room temperature (kept in the dark). Cells were then incubated with FITC-SNA (Vector Laboratories) for 15 min at room temperature (kept in the dark). All slides were mounted in EverBrite Hardset mounting medium with DAPI (Biotium, Hayward, CA, USA). The fluorescent signals from EGFR and FITC-SNA were recorded and analyzed with TCS SP5 Confocal Spectral Microscope Imaging System (Leica Microsystems, Wetzlar, Germany). The figures shown are representative of four fields for each experimental condition. Nuclei were defined with DAPI staining.

Paraffin embedded sections (10 µm) were deparaffinized in CitriSolv (Fisher Scientific; Fairlawn, NJ) and rehydrated through a series of graded ethanol to distilled water steps. Antigen retrieval (10 mM sodium citrate, pH 6.0) and blocking (1:500 dilution; 100 µL rat serum:50 mL TRIS-buffered saline, 0.1% Tween 20 (TBST) for 1 h) was undertaken prior to sequential washes (TBST) and primary antibody incubation. Primary antibodies (VEGF [clone # VG1 – Invitrogen Cat# MA5-12184, used at 1:100], CD31 [clone #390 – Invitrogen Cat# 14-0311-82, used at 1:100], Col2 [clone # II-II6B3 – DSHB, Iowa, used at 1:50], αSMA [clone # 1A4 – ABCAM Cat# ab7817, used at 1:100] and PRG4 [clone # 9G3 – Millipore, Cat# MABT401, used at 1:100]) were directly conjugated to fluorophores using the DyLight® 488, 550 or 650 Conjugation Kit [ABCAM]. All slides were mounted using EverBrite™ Hardset Mounting Medium with DAPI (Biotium) for nuclear counterstaining and coverslipped. Slides were imaged using a Plan-Apochromat objective (10x/0.8 M27) on an Axio Scan.Z1 Slide Scanner microscope (Carl Zeiss); DAPI (353 nm/465 nm), EGFP/FITC/Alexa Fluor 488 (493 nm/517 nm), R-PE (565 nm/576 nm), and APC (650 nm/660 nm) specific filters were employed. Isotype controls for Alexa Fluor 488, 550 or 650 demonstrated little to no reactivity.

Exponentially growing primary colorectal cancer cells and HT-29 were seeded on sterilized cover glasses (Paul Marienfeld, LaudaKönigshofen, Germany). After treatment of NDAT (0.1 μM) or LY294002 (10 μM) for 24 h, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min and then permeabilized in 0.06% Triton X-100 for 30 min. Cells were incubated with monoclonal rabbit anti-PD-L1 antibody, followed by an Alexa-647-labeled goat anti-rabbit antibody (Abcam, Cambridge, MA, USA) and mounted in EverBrite Hardset mounting medium with DAPI (Biotium, Fremont, CA). The fluorescent signals were recorded and analyzed with the TCS SP5 Confocal Spectral Microscope Imaging System (Leica Microsystems). The figures shown are representative of at least four fields for each experimental condition.

Immunofluorescence in HCC cells was performed as previously described.17 Briefly, cells were fixed in 4% paraformaldehyde for 20 minutes and permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich) for 15 minutes. Primary antibodies were from Abcam (anti‐gamma H2A.X (phospho S139), anti‐CDKN2A/p16INK4a, anti‐p21 [CP74]; diluted 1:500), Santa Cruz Biotechnology (anti‐p53 (FL‐393); diluted 1:1000) and R&D Systems (anti‐DPPIV/CD26; diluted 1:1000). The staining was developed using Alexa Fluor‐conjugated secondary antibodies (488, 555 or 633, ThermoFisher Scientific). Cover glasses were placed into EverBrite Hardset Mounting Medium with DAPI (Biotium), and images were acquired using an Axio scan Z.1 or LSM 7 DUO system (Zeiss) confocal microscope.