Prolongtm gold antifade reagent with dapi

TUNEL immunostaining was used to label dead cells in 5 µm thick formalin-fixed, paraffin-embedded lung tissues using the In Situ Cell Death Detection Kit, TMR red (Roche, 12156792910) to measure the double-stranded cleavage of DNA according to the manufacturer’s instructions. Sections were deparaffinized by incubating them in a 60°C incubator for 1 h, followed by 15 min of immersion in xylene with two changes. Rehydration was done with a graded series of ethanol (100%, 90%, 70%, and 50% (v/v) ethanol) twice for 10 min. Sections were washed with deionized water for 5 min two times, then stained with TUNEL according to the manufacturer’s instructions. After that, the sections were washed twice in PBS. Finally, the nuclei were stained with 4′,6-diamidino-2-phenylindole-DAPI by mounting them in ProlongTM gold antifade reagent with DAPI (Invitrogen, P36931) and stored in the dark. An Evos FL Auto 2 microscope (Invitrogen) was used to examine stained sections, and images of six random fields were examined at an objective 10× magnification for analysis. ImageJ (https://imagej.nih.gov/ij/) was used to measure TUNEL positivity lung tissue images. The mean fluorescence within a region of interest (ROI) was measured and normalized by the intensity of DAPI fluorescence.

Tumors were harvested from sacrificed mice 1 h after ICG or ICG-HSA injection and embedded in optimal cutting temperature (OCT) compound (Leica biosystems, Wetzlar, Hesse, Germany). After freezing at −80 °C for 24 h, the specimens were cut into 8 μm sections. Frozen tumor slides were thawed for 5 min at RT, followed by permeabilization of samples with acetone at −20 °C for 10 min and washing the slides three times with PBS for 5 min. After the permeabilization step, the samples were blocked with 5% BSA in PBS for 1 h at RT. After blocking, the samples were treated with anti-SPARC primary antibody (#AF941; R&D systems, Minnneapolis, MN, USA; diluted 1:10) in 0.5% BSA and incubated at 4 °C overnight. On the following day, the samples were incubated with Alexa Fluor^TM488 donkey anti-goat IgG secondary antibody (A11055; Invitrogen; diluted 1:400) in 0.5% BSA for 2 h at RT. After incubation, the samples were washed three times with PBS for 5 min and mounted with ProLong^TM Gold antifade reagent with DAPI (Invitrogen). Fluorescence signals were obtained by confocal laser scanning microscope (Leica TCS SP8; Leica).

Isolated primary murine PDAC cells and MIA PaCa-2 cells were seeded on fibronectin-coated 20 mm diameter cover glasses in 12-well plates (1×10⁵ cells/well) and cultured in 37 ℃ for 24 h. Cells were then fixed with 4% PFA for 30 min, permeabilized with 0.1% Triton X-100 in 1× PBS for 10 min, blocked with 5% BSA for 30 min and incubated with anti-Kindlin-2 (clone 3A3, Millipore, MAB2617, 1:5000), anti-DDX3X (Proteintech, 11115-1-AP, 1:1000), anti-c-Myc (Abcam, ab32072, 1:100) antibodies overnight at 4 ℃. Cells were then washed with 1×PBS three times and incubated with secondary antibodies (Alex fluor, 1:500) for 30 min at room temperature. Cells were mounted with ProLong^TM Gold antifade reagent with DAPI (Invitrogen, P36941) and visualized by Zeiss LSM980 with an Airyscan2 imaging system.

HepG2 cells were seeded on sterile glass coverslips inside 24-well culture plates at 6 × 10⁴ cells/well density overnight and treated with specific treatments for 24 h. Cells were fixed with 4% paraformaldehyde and washed with phosphate-buffered saline with Tween 20 (PBS-T) before being blocked with 1% BSA containing glycine prepared in PBS for 1 hour at room temperature. Then, cells were incubated with specific primary antibodies that were prepared in 1% BSA (1:200 dilution for PD-L1 antibody) for overnight at 4 °C. After being washed with PBS-T three times, cells were incubated with fluorochrome-conjugated secondary antibody, anti-rabbit IgG-FITC (Alexa Fluor 488), in 1:1000 dilution for 1 h at room temperature in the dark. Stained coverslips were mounted on the glass slides with a drop of mounting media containing DAPI: ProLongTM Gold antifade reagent with DAPI (Invitrogen, Thermo Fisher Scientific). Slides were photographed under a fluorescence microscope (Scope A1, Carl Zeiss). Images were analyzed using ImageJ software.

IEC-6 cells were cultured in standard condition for 12 h before the experiment. Cells were fixed with 4% paraformaldehyde (SenBeiJia, Nanjing, China) for 20 min. Then cells were incubated in 0.2% Triton X-100 (Solarbio) for 5 min. Next, the nonspecific protein binding sites were blocked with 3% bovine serum albumin (BSA)-phosphate-buffered saline ((PBS); ZSGB-BIO, Beijing, China) at 37 °C for 1 h. Then cells were incubated overnight with related primary antibody at 4 °C. After washing with PBS for 15 min, cells were incubated for 1 h at 37 °C with an anti-rabbit secondary antibody (SA00003-2, Proteintech). Cells were then treated with prolong^TM gold antifade reagent with DAPI (Invitrogen) and were stored from night. Images were verified using the confocal microscope (FV1000PME, Japan). Colon sections were processed in the same way as above, and the images were verified by fluorescence microscope. Furthermore, the inflammation caused by DSS or TNBS was assessed by hematoxylin and eosin (HE).

Dispersed islet cells were washed twice in PBS, resuspended in PBS, and centrifuged onto microscope slides using a Shandon Cytospin II (ThermoFisher Scientific). Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 30 min and blocked using 1% BSA in PBS with 0.2% Tween (PBST). Primary antibodies to insulin and EMCV capsid were used at 1:1000 in PBST for 1 h. Secondary antibodies Cy3-conjugated donkey anti-guinea pig and AF-488-conjugated donkey anti-rabbit were used at 1:1000 in PBST in a dark, humidified chamber for 1 h. ProLong^TM Gold Antifade Reagent with DAPI (Invitrogen) was used to preserve fluorescent signal and for nuclear staining. Images were captured using a Nikon eclipse 90i confocal microscope.