Leica photomicroscope

The histopathologic examination was carried out on pancreas specimens (in 10% formalin) using light microscopy. Then, samples were processed to obtain 5 µm thick paraffin sections, followed by staining with hematoxylin and eosin (E) and observation under a Leica photomicroscope.

Antigen retrieval was performed on deparaffinized sections using the Target Retrieval Solution (Dako, USA). After quenching in Dako REAL™ Peroxidase Blocking Solution (Dako, USA), the slides were incubated with primary antibodies against Nrf2 (Affinity, China), HO-1 (Affinity, China), MMP-3 (Proteintech, China), and MMP-13 (Proteintech, China) overnight at 4°C. Following three PBS washes, the sections were incubated with the corresponding secondary antibody for 50 min followed by an examination on the liquid diaminobenzidine (DAB) + substrate-chromogen system. Leica photomicroscope (Leica, Germany) was used to capture the images.

The expression of ASIC3 in retrogradely labelled DRG neurons was assessed by immunofluorescence. Tissue sections were first incubated with an anti-ASIC3 antibody from the rabbit (from Alomone, diluted at 1:300) overnight at 4°C. This was followed by 1 h incubation with a goat anti-rabbit secondary antibody conjugated with Alexa-488 (from Invitrogen, diluted at 1:300), three washes in PBS and coversliped using glycerol/DABCO mounting media. Pictures were taken with a Leica photomicroscope at x10 magnification.

BALB/c athymic nude mice (6 weeks old male) were kept in a specific pathogen-free environment. All animal experiments were conducted at Laboratory Animal Center of Institute of Radiation Medicine, the Chinese Academy of Medical Sciences in accordance with the Institutional Animal Care and Use Committee guidelines. Mice were injected subcutaneously (s.c.) in the flank with 1×10⁷ A549 cells. When subcutaneous tumors grew to 30 to 50 mm³, the mice were randomly divided into four groups, with six mice each. For exosome treatment, 2 μg of exosomes such as Exo, Exo-c-Jun-KO, and those treated with miR-494 agomir or agomir NC was injected intratumorally every other day for 14 days. Tumor volume was measured every other day. Mice were sacrificed by cervical dislocation 16 days after treatment, and tumor specimens were fixed with 4% formaldehyde, embedded in paraffin, and subjected to routine histological experiment. Immunohistochemistry (IHC) was carried out on 5-μm sections to visualize cells by using Ki-67, CD31, α-smooth muscle actin (α-SMA) antibodies. Six field images were collected using a Leica photomicroscope from three biopsies per specimen. Staining intensity was analyzed by Image-pro plus software.