Paraformaldehyde (pfa)

To obtain histological profiles of pancreas, pancreatic tissues were fixed in 10% formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by microscopy. For immunofluorescence staining, pancreatic tissues were fixed in 4% paraformaldehyde (Cat#G1101, Servicebio), OCT (Cat#4583, Tissue-Tek) embedded, and sectioned. Sections were permeabilized in PBS containing 0.3% Triton X-100 (Cat#T8787, Sigma) and blocked in QuickBlock™ Blocking Buffer (Cat#P0260, Beyotime) prior to incubation with primary antibody against insulin (Cat#4590 S, CST, 1:100 dilution in blocking buffer) or Glut2 (Cat#sc-518022, C-10, Santa Cruz, 1:200 dilution in blocking buffer). Sections were then washed and incubated with Alexa Fluor 594-conjugated Donkey anti-Rabbit IgG (H + L) (Cat#711-585-152, Jackson, 1:200 dilution in blocking buffer) or Alexa Fluor 488-conjugated Donkey anti-Mouse IgG (H + L) (Cat#715-545-150, Jackson, 1:200 dilution in blocking buffer). After washing, sections were incubated with DAPI (Cat#H-1200, Vectorlabs) to stain the nuclear. Images were acquired on a confocal laser scanning microscope using Zen software (v2.3, Carl Zeiss) and analyzed using Image pro software (v6.0, Media Cybernetics).

For radiographic analysis, the hind limbs were imaged on a multimodality in vivo imaging system (Kodak Company, Rochester, NY, USA) shortly before the rats were sacrificed. The exposure time was 10 s, and the f-stop factor was 2.5.
For histopathological analysis, the protocol was performed as previously described [31 (link)]. Briefly, the fresh ankle joints were fixed with 4% paraformaldehyde (Servicebio, Wuhan, China) and then decalcified with 10% ethylene diamine tetraacetic acid (EDTA, Servicebio, Wuhan, China) at room temperature for 40 days. The processed joints were dehydrated, embedded, and sectioned at 4 μm thickness in a sagittal plane. Sections were then stained with hematoxylin-eosin (HE) for assessing EA's effect on the histopathological damage in joints. Images were acquired and observed under an upright light microscope (Nikon Inc., Tokyo, Japan). HE staining score was evaluated by an investigator who was blinded to the experimental protocol. The following morphological criteria were considered as follows [32 (link)]: score 0, no damage; score 1, edema; score 2, presence of inflammatory cells; score 3, bone resorption.

Cell migration and invasion ability were examined by Corning transwell insert chambers (8 mm pore size, Corning). The transwell chamber was placed into the 24-well culture plate, the chamber was called the upper chamber, and the culture plate was called the lower chamber. In the migration assay, 2 × 10⁵ cells were suspended in 100 μl FBS-free medium and were added to the upper chamber. 600 μl RPMI 1640 with 20% FBS were seeded in the lower compartment of the chamber and the cells were incubated in an environment of 37°C and 5% CO₂. After 24 h, 4% paraformaldehyde (Servicebio, China) was used for fixation. Afterwards, 0.5% crystal violet was dyed for 10 min and the membrane was washed with PBS. The migrated cells on the lower surface were photographed randomly with an Olympus IX51 inverted microscope in five visual fields (× 200 magnification) and the migrated cells were quantified using Image J software. For invasion assay, the upper compartment was precoated with one hundred microliters of matrigel and 5 × 10⁵ cells per invasion well were added to the upper chamber. After incubating for 48 h, the rest of the protocol was similar to the migration assay. Each assay was performed at least three times.

The clinical PC and adjacent tissues were fixed with paraformaldehyde (Servicebio, Wuhan, China), embedded in paraffin (Boster, Wuhan, China), and sectioned into 4 μm thickness. After dewaxing, rehydration, and antigen retrieval with sodium citrate (Servicebio, Wuhan, China), tumor samples were blocked using H₂O₂ and 5% bull serum albumin (Servicebio, Wuhan, China) for 30 min at room temperature. The specimens were incubated with primary anti-TRA2A (1:100), KI67 (1:100), anti-PCNA (1:100), and anti-HIF1α (1:100) antibodies for 12 h and horseradish peroxidase-conjugated secondary antibodies (Zsjqbio.biogo; Beijing, China). Next, DAB was used to identify the antigen-antibody complex, and the samples were stained with hematoxylin to visualize the nucleus.