Alexa fluor 488 and 594 antibodies

Cells grown on 13 mm coverslips were fixed in 4% paraformaldehyde (w/v) in PBS for 15 min, prior to permeabilisation in 0.5% Triton X-100 (v/v) in PBS for 15 min. Cells were then blocked in blocking buffer for 1 h, prior to 1 h labelling with primary antibody at 1 : 500 dilution (anti-HA (Y-11) (Santa Cruz) or anti-Flag antibody). Cells were washed three times before being stained with secondary antibody for 1 h (anti-mouse and anti-rabbit Alexa Fluor 488 and 594 antibodies (Thermo Fisher)). Cells were washed three more times and then mounted on glass slides using Vectorshield mounting media containing DAPI (Vectorlabs; Peterborough, UK). Images were collected using Openlab5 software (Improvision; Coventry, UK) and an Olympus BX60 fluorescent microscope fitted with a Hamamatsu C4742-95 camera.

For Immunofluorescence studies, cells were grown on glass coverslips for 2 days, and then cells were washed with 1× PBS three times, then fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were then passed through frozen methanol for 10 min at 20 °C and blocked in 5% BSA for 30 min. slides Incubated with primary antibody (1:100–1:200) in 5% BSA, overnight at 4 °C, and then incubated secondary antibody (anti-mouse and anti-rabbit Alexa Fluor 488 and 594 antibodies (Thermo Fisher) (1:100) in 5% BSA for 60 min at room temperature. An Olympus FV1000 confocal microscope with a ×60 objective was used for taking images. The co-localization was analyzed with image pro plus software.

We used immunofluorescence double-staining to systematically analyze the collected PDAC specimens. Consecutive 3 μm sections from formalin-fixed and paraffin-embedded pancreatic tissue were incubated overnight with the corresponding antibodies (CK19-Anti-KRT19, rabbit, Sigma-Aldrich, Munich, Germany, 1:400; Mucin1-Anti-MUC1, mouse, Santa Cruz Biontechnology, Dallas, TX, USA, 1:400; Insulin-Anti-Insulin, mouse, HyTest Ltd., Turku, Finland, 1:500; and Glucagon-Anti-glucagon, rabbit, Cell Signaling Technology, Danvers, MA, USA, 1:400) after antigen retrieval via incubation in citrate buffer in a humid chamber at 4 °C. Finally, staining was detected with fluorescein-conjugated secondary antibodies (Alexa Fluor 488 and 594 antibodies, Life Technologies, Carlsbad, CA, USA, 1:300). Nuclei were stained using DAPI. Islet invasion patterns were analyzed by two independent observers (U.C. and Y.C.E) after imaging using the BioRevo BZ-9000 platform (Keyence, Osaka, Japan).

AV samples were embedded in optimal cutting temperature compound (VWR, USA) and cut into 7 μm cryosections. All sections were fixed in 4% paraformaldehyde prior to staining. We performed blocking with the applicable serum followed by primary antibody incubation (Table S3).
Biotin-labeled secondary antibodies (1:200; Vector Laboratories, USA) and Alexa Fluor 488 and 594 antibodies (1:200; Life Technologies, USA) were used. Sections were embedded in 4,6-diamidino-2-phenylindole mounting medium (Vector Laboratories). We used streptavidin-labeled horseradish peroxidase solution (Dako, USA) and 3-amino-9-ethylcarbazole (Dako, USA) solution for staining development. Immunohistochemistry samples were examined with an Eclipse 80i microscope (Nikon, USA). Immunofluorescence sections were analyzed with a Nikon A1 confocal microscope (Nikon, USA).

Following nPM treatment and euthanasia, the animals’ heads were de-skinned, fixed overnight in 10% neutral buffered formalin at 4°C, and decalcified (Shandon’s TBD-1 Decalcifier; Thermo Scientific, Waltham, MA); then, the samples were cryoprotected by submersion in a 10–30% sucrose/PBS pH 7.4 gradient. The heads were embedded in optimal cutting temperature (OCT) compound for cryostat sectioning. For the OE sections, brain tissue was removed posterior to the OB. Tissue sections (18 μm) on glass slides were permeablized with 1% NP-40/PBS and blocked with 5% BSA. Primary antibodies for markers of olfactory sensory neurons [OMP (1:100, goat; Santa Cruz) and NeuN (1:100, mouse; Millipore)], astrocytes [GFAP (1:400, mouse; Sigma)], microglia [Iba1 (1:200, rabbit; Wako)], and oxidative stress [4-HNE (1:100, rabbit; Millipore) and 3-NT (1:100, rabbit; Abcam)] were added at 21–23°C and incubated overnight. Immunofluorescence was visualized using Alexa Fluor 488 and 594 antibodies (1:400, goat; Molecular Probes), 4’,6-diamidino-2-phenylindole (DAPI; Vector Labs), and HRP (1:400, goat; Jackson) + 3,3′-diaminobenzidine (DAB).