Cd163

For immunofluorescent double staining of M1 (F4/80⁺ CD86⁺) and M2 (F4/80⁺ CD206⁺), paraffin‐embedded spleen sections were processed with deparaffinized, rehydrated and antigen retrieved. After fixing in 4 % paraformaldehyde for M1 (CD68⁺ CD86⁺) and M2 (CD68⁺ CD163⁺), THP‐1 cells were permeabilized with 1 % Triton X‐100, then blocked and incubated overnight with primary antibodies at 4°C, followed by staining with Alexa Fluor‐conjugated secondary antibody (1:500). The following primary antibodies were used: CD68, CD163, F4/80, CD86 (Cell Signaling Technology, USA) and CD206 (Abcam, USA). Cells were imaged with a Nikon Eclipse 800 upright epifluorescence microscope (Nikon Instruments, NY).

Cells and purified exosomes were lysed in RIPA buffer (Sigma) containing protease inhibitor cocktail (Roche) and total protein concentration was tested using bicinchoninic acid protein determination kit (Sigma). Aliquots of 25 µg of proteins were separated on SDS/PAGE gel and transferred to a poly(vinylidene difluoride) membrane. Then the membranes were blocked with 5% BSA, incubated with primary antibodies against CD163 (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), TSG101 (1 : 2000; Abcam), E‐cadherin (1 : 1500; Cell Signaling Technology), N‐cadherin (1 : 1000; Cell Signaling Technology), MMP‐9 (1 : 2000; Cell Signaling Technology), MMP‐2 (1 : 2000; Cell Signaling Technology), slug (1 : 3000; Cell Signaling Technology), VEGFR (1 : 1000; Cell Signaling Technology), TIMP3 (1 : 1000; Cell Signaling Technology), GADPH (1 : 5000; Cell Signaling Technology) or β‐actin (1 : 5000; Cell Signaling Technology) overnight at 4 °C. The blots were washed, incubated with HRP‐conjugated secondary antibody (1 : 5000; Cell Signaling Technology) at room temperature for 1 h, and proteins were detected using Pierce ECL Western Blotting Substrate under an Odyssey infrared scanner (LI‐COR Biosciences Inc., Lincoln, NE, USA). Western blot bands were analyzed with NIH imagej software.

Following fixation, gels were dissected into 2mm × 2mm pieces and placed into a 96 well plate. Gentle agitation was provided during staining via a platform shaker at 23°C. Gel pieces were washed in PBS for 30 minutes. They were then permeated with 0.1% TritonX-100 in PBS (ThermoFisher, Cat:85111) for 30 minutes. Gels were then blocked with PowerBlock (BioGenex, Cat: HK085-GP) for 1 hour. Following blocking, primary antibody was made up in a 1:1 solution of 0.1% TritonX-100 and PowerBlock. Primary staining lasted 1 hour, followed by two 15-minute washes with 0.025% Tween 20 in PBS (Sigma, Cat: P1379) and a single 15-minute wash in PBS. Secondary antibody containing Alex Fluor 488, 594, or 647 against the primary antibody was added to a 1:1 solution of 0.1% TritonX-100 and PowerBlock. Secondary antibody staining lasted 45 minutes, followed by two 30-minute washes in 0.025% Tween 20 in PBS (Sigma, Cat: P1379) and a single 30-minute wash in PBS. Stained gel pieces were mounted on a slide with Fluoromount-G mounting solution with DAPI (ThermoFisher Cat:00495952). Fluorescent images were taken with an LSM880 inverted confocal microscope.
The following primary antibodies were used for staining gels: CD45 (Abcam, Cat: ab8216), CD163 (Cat: PA5–78961), and COL1 (Cell Signaling, Cat: 72026S).

Assays were performed as previously reported [20 (link)]. In brief, for the IF experiment, cells were incubated with primary antibodies against CCL20 (1:200, #ab9829) (Abcam) at 4 °C overnight, then incubated with the fluorescent secondary antibody Alexa Fluor 594-conjugated goat anti-mouse IgG (Cell Signaling Technology) and imaged using a fluorescence microscope (DM5000B, Leica). For IHC, the paraffin sections were incubated with antibodies against CCL20 (1:200, #ab9829) (Abcam). Intensity scores were recorded as: 0 (no staining), 1 (weakly staining, light yellow), 2 (moderately staining, yellowish brown), and 3 (strongly staining, brown). In addition, we also detected the expression of ki67 (1:1000, #9449), CD68 (1:200, #97778), CD163 (1:400, #93498), CD206 (1:200, #24595) and CD31 (1:100, #77699) (Cell Signaling Technology) in PCa tissue or xenograft tissue. Images were observed under an Olympus multifunction microscope (Olympus BX51, Tokyo, Japan). All evaluations were performed by three independent senior pathologists using the same microscope.

Antibodies against TNFα, IL-6, CD68, CD163, MCP-1, F4/80-FITC, CD11c-PE, and CD206-AF488 were purchased from Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primers (Table 1) of TNFα, MCP-1, IL-4, IL-13 and 18S were purchased from Applied Biosystems. All other reagents were obtained from Sigma (St. Louis, MO, USA). Tissue levels of the adhesion molecules ICAM-1, VCAM-1 and MCP-1 were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available ELISA kits (BD Bioscience, San Jose, CA, USA).

Immunohistochemistry (IHC) was conducted on 8 μm thickness sectioned paraffin slides. Following incubation for one hour at 50 °C, slides were washed twice in Tween 20 (Sigma-Aldrich^™, St. Louis, MO) followed by one wash in PBS. Slides were then blocked for 1 hour with Power Block (Biogenex^™, Fremont, CA) prior to addition of the following primary antibodies: CD45 (Abcam, Cat: ab8216), CD163 (Cat: PA5–78961), CD93 (Cat: PA5–52930), CAV1 (Cat: ab214448), CD40 (Cat: 2246639), COL1 (Cell Signaling, Cat: 72026S). Slides were then incubated for 1 hour with Alexa Fluor 488, 594, or 647-conjugated anti-rabbit, anti-rat, or anti-mouse antibodies (Invitrogen, Waltham, MA). Finally, slides were mounted in Fluoromount-G mounting solution with DAPI (ThermoFisher Scientific^™, Waltham, MA). Images were acquired with a LSM880 inverted confocal, Airyscan, AiryscanFAST, GaAsP detector upright confocal microscope.