Fluorescein isothiocyanate (fitc)

Fresh tissue specimens, which were embedded in OCT compound and frozen in acetone-dry ice mixture, were cut at a thickness of 3 μm on a cryostat. The frozen sections were fixed in 1:1 of acetone and methanol, and then blocked with 10% goat serum in 0.01 mol/L phosphate-buffered saline.
Staining of CD31, ADPN, AdipoR1, AdipoR2, and T-cadherin (cadherin-13) was performed by indirect immunofluorescence using the primary monoclonal or polyclonal antibodies listed in S1 Table.
Anti-mouse, rabbit, or goat IgG polyclonal antibodies conjugated with fluorescein isothiocyanate (MP Biomedicals), Alexa Fluor 455, or Alexa Fluor 488 (Thermo Fisher Scientific) were used as secondary antibodies, and their signals were visualized using a BX51/DP71 fluorescence microscope/CCD camera (Olympus).

The preparation of aluminum hydroxide was done following the protocol reported previously (Reithofer et al., 2020 (link)). In brief, 5% NaOH and 5% Al₂(SO₄)₃ were sterilized by passing through a 0.22-μm filter. After being heated at 60°C for 30 min, two volumes of 5% NaOH and five volumes of 5% Al₂(SO₄)₃ were mixed with stirring, followed by centrifugation at 10,000 g for 5 min. After being washed twice with sterile phosphate-buffered saline (PBS), the mixture was suspended in L-15 medium (HyClone, Logan, UT, USA) to give 8 mg ml⁻¹. For fluorescence-dyed Alum formulation, a total of 10 mg of fluorescein isothiocyanate (FITC) (MP Biomedicals, Solon, OH, USA) was added to Alum suspension and incubated for 24 h in the dark at room temperature on a rotator. Free FITC was removed from FITC-Alum by 12,000 g of centrifugation for 10 min and then washed 10 times or until no fluorescence was present in the supernatant. The pellets were resuspended in PBS and used to stimulate peritoneal cells. For OVA/Alum formulation, Alum was mixed with an equal volume of 10 mg ml⁻¹ of OVA (grade V; Sigma, Poole, UK) and incubated at room temperature for 20 min. After centrifugation at 14,000 g for 10 min, sedimented OVA/Alum was resuspended in PBS. For OVA/oil formulation, OVA was emulsified with Freund’s incomplete adjuvant (FIA; Sigma, St. Louis, MO, USA) at equal volume.