Pv 9000

Immunohistochemistry was performed by two-step method according to the instructions (PV-9000; ZSGB-BIO, Beijing, China). Pancreatic cancer samples were fixed in 10% formalin, embedded in paraffin, and processed into 5-µm sequential sections. The samples were de-waxed with ethanol and blocked to inhibit the endogenous peroxidase activity. After this, samples were heated in a microwave for antigen retrieval, cooled to room temperature, and blocked using goat serum for 30 min at 37 °C. The samples were incubated overnight at 4 °C with rabbit anti-ALOX5 (ab169755), anti-ALOX12 (ab211506), and anti-CISD1 (ab203096) (Abcam, USA) (1:200), followed by incubation with horseradish peroxidase-coupled goat anti-rabbit secondary antibody (PV-9000; ZSGB-BIO, Beijing, China) at 37 °C for 30 min. The samples were then stained with 3,3′-Diaminobenzidine (DAB). Cell nuclei were stained blue with hematoxylin. The sections were then dehydrated, cleared with xylene, and mounted. ALOX5, ALOX12, and CISD1 expressions were determined by immunohistochemistry (IHC) using the streptavidin peroxidase method, with adjacent tissues serving as the controls. The experimental procedure was performed as per the manufacturer’s instructions. Image-Pro Plus 6.0 Software (Media Cybernetics, USA) was used to analyze protein expression and perform statistical analysis of the results obtained by IHC.

Formalin‐fixed paraffin‐embedded 5‐μm tissue sections were incubated at 60°C for 30 minutes and then deparaffinized in xylenes, rehydrated through a graded series of alcohol concentrations for 5 minutes and rinsed by water for 2 minutes. After antigen retrieval by 1mM Tris‐EDTA (pH = 8.0) was performed, all sections were rinsed with phosphate buffer saline (PBS) for three times and left to block at room temperature in 3% H₂O₂‐methanol for 10 minutes, followed by the coating of antibodies at room temperature for overnight at 4℃. Afterwards, the sections were rinsed three times in 0.1% PBST, incubated with an Enhancer buffer (PV‐9000, Zsbio, Beijing, China) for 20 minutes and incubated with enzyme‐labelled anti‐mouse/rabbit secondary antibodies (PV‐9000, Zsbio) for 30 minutes. Subsequently, the proteins were subjected to diaminobenzidine (DAB) development and left to counterstain with haematoxylin for 1 minute. After the differentiation and addition of blue coloration, the sections were dehydrated by gradient concentration ethanol, transparentized and sealed by neutral resins. Immunohistochemistry images were obtained using an upright microscope (BX53, OLYMPUS, Japan) and analysed and scored by experienced pathologists. Primary antibodies used: KDM5A (1:400, Abcam, Cambridge, UK, ab217292) and CD31 (1:400, Abcam, ab9498).

H&E staining was carried out as the standard protocol described. Paraffin sections were dewaxed, hydrated, and stained with hematoxylin and eosin, respectively, and then dehydrated and mounted. IHC was performed to measure the expression of UCP1 protein in mouse adipose tissues as described previously [47 (link)]. Section was dewaxed and hydrated, followed by antigen retrieval. Endogenous peroxidase was blocked by 3% hydrogen peroxide solution (PV-9000, ZSGB-Bio, Beijing, China). The section was incubated with the blocking goat serum (C0265, Beyotime, Shanghai, China) for 15 min and immunostained with anti-UCP1 antibody (ab10983, Abcam, Cambridge, UK) overnight at 4 °C. After three washes with PBS, the slides were stained with horseradish peroxidase-conjugated anti-rabbit IgG (PV-9000, ZSGB-Bio, Beijing, China) and DAB Chromogenic Kit (ZLI-9018, ZSGB-BIO, Beijing, China), followed by counterstaining with hematoxylin. The full section was scanned by using the Panoramic Scanning Microscope-VS120 (Olympus Life Science, Tokyo, Japan). Cell size and the density of UCP1-positive staining (brownish yellow) was analyzed by Image J.

The formalin fixation and paraffin embedding tissue sections were dried overnight at 60 ℃, deparaffinized in xylene for 30 min, rehydrated through a gradient alcohol solution, boiled for 15 min in antigen retrieval solution containing EDTA (ZLI-9066, Zsgb-Bio, China), treated for 10 min with endogenous peroxidase blocker (PV-9000, Zsgb-Bio, China), and incubated for 10 min with normal goat serum (ZLI-9022, Zsgb-Bio, China). The sections were incubated overnight at 4 ℃ with a rabbit monoclonal primary antibody against RARRES2 (10216-1-AP, Proteintech, China), then incubated for 25 min with biotinylated secondary antibodies (PV-9000, Zsgb-Bio, China). Color development was performed with 3,3N-diaminobenzidine tertrahydrochloride (ZLI-9019, Zsgb-Bio, China). The sections were counterstained with hematoxylin, mounted by slides with neutral balsam, and scanned by Aperio patholgy scanner (Leica, Germany).