F ab 2 fragment

Double immunofluorescence staining for human colitis tissue of UC patients, normal colon tissue of healthy volunteers, and murine colitis tissue was performed. Frozen sections were treated with citrate buffer (pH 6.0) for 10 min at 100 °C for antigen retrieval and then blocked with blocking solution for 30 min at room temperature. Sections were incubated with a mixture of 2 primary antibodies overnight at 4 °C. The sections were then incubated with a mixture of 2 secondary antibodies for 1 h at room temperature. The following secondary antibodies were applied: Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L), F(ab’)₂ Fragment (#4408, Cell Signaling Technology, Danvers, MA), Alexa Fluor 555-conjugated goat anti-rabbit IgG (H + L), F(ab’)₂ Fragment (#4413, Cell Signaling Technology), and Alexa Fluor 488-confugated goat anti-rat IgG (H + L) (#4416, Cell Signaling Technology). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). The stained sections were observed using a BZ-X710 fluorescence microscope (Keyence, Osaka, JPN).

Briefly, brain slices were preincubated with 0.1% Triton X-100 (v/v) in 0.01 M PBS (pH 7.4) for 15 minutes. After blocking with 10% normal goat serum (Sigma-Aldrich), slides were incubated with primary antibodies as follows: rabbit anti-GFAP, rabbit anti-myelin basic protein (MBP), and rabbit anti-IBA1 (1 : 400; Abcam, Cambridge, MA, USA). After incubated overnight at 4°C and rinsed in 0.01 M PBS (3 × 5 minutes), tissue slices were incubated with Alexa Fluor® 488 conjugated goat anti-rabbit IgG (H + L), F(ab')2 Fragment or Alexa Fluor® 594 conjugated goat anti-mouse IgG (H + L), F(ab')2 Fragment (1 : 1000; Cell Signaling Technology) in 0.01 M PBS for 1 hour at room temperature. If necessary, the sections were counterstained for nuclei with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1 : 1000; Roche, Mannheim, Germany), and then slides were mounted in the ProLong® Gold antifade reagent (Thermo Fisher Scientific, USA) prior to imaging. The immunofluorescence intensity was analyzed with Image J, v1.8.0.

Rabbit IgG anti-phospho-PKA substrate (RRXS*/T*) (100G7E), 1:1000 (#9621); anti-Akt (pan), 1:2000 (#3673); anti-phospho-Akt (T308), 1:1000 (#2965); phospho-Akt (S473), 1:2000 (#4060); anti-ERK1/2, 1:2000 (#4695); phospho-ERK1/2 (Thr202/Tyr204), 1:2000 (#4370); Alexa Fluor® 555-conjugated goat IgG anti-mouse IgG (H+L) and F(ab')2 Fragment were purchased from Cell Signaling Technology (Danvers, MA). Mouse IgG anti-human APJ (APLNR) was obtained from R&D systems (Minneapolis, MN), 1:1000 (#MAB8561). Mouse IgG anti-GAPDH, 1:10000 (#014-25524) and anti-FLAG were from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), I.F. 1:200, W.B. 1:1000 I.P. 1:500 (#014-22383). Rabbit IgG anti-myelin protein zero was from Abcam (Cambridge, UK), 1:1000 (#ab31851).