L a b solution

Paraffin-embedded brain tissue samples were sectioned (5 μm) and dewaxed, followed by antigen retrieval using L.A.B solution (Polyscience, Inc) for 15 minutes. Soon afterwards, the tissue samples were incubated with mouse anti-Aβ (1:200; Santa Cruz Biotechnology) or rabbit anti-synaptophysin (SYP, 1:200; Sigma) at 4 °C overnight. After the tissue samples were rinsed with PB (0.01 mmol/L) and incubated in secondary antibodies, they were immunostained in 0.025% DAB for immunohistochemical detection. Slides were sealed with resin to be viewed under the microscope. For double immunofluorescence, frozen sections (10 μm) were incubated with mouse anti-Aβ (1: 200; Santa Cruz Biotechnology) and rabbit anti-glial fibrillary acidic protein (GFAP, 1:100; Sigma) or mouse anti-Aβ (1:200; Santa Cruz Biotechnology) and rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba1, 1:100; Sigma) primary antibodies overnight at 4 °C. Secondary antibodies, donkey anti-mouse IgG conjugated with fluorescein isothiocyanate (1:200; Jackson ImmunoResearch Laboratories) and Texas-Red donkey anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories), were used for 2 hours at room temperature, followed by DAPI for 5 minutes. The images were acquired using a confocal laser scanning microscope (SP8, Leica).

The expression of CD31 was assessed by immunohistochemical staining of formalin-fixed and paraffin-embedded vascular organoids in a fibrin gel with CMF-20 µm. To liberate antibody-binding sites using L.A.B. solution (Polysciences Inc., Warrington, PA, USA) and to block endogenous peroxidase activity, blocking was performed for 20 min at room temperature using a VECTASTAIN Elite ABC kit (mouse IgG; #PK-6102; Vector Laboratories, Burlingame, CA, USA). Then, 3.5-µm thick sections were incubated overnight at 4 °C with the mouse monoclonal anti-CD31 antibody (dilution, 1:200; Wako, M0823, Osaka, Japan). Hematoxylin was used for nuclear staining for 1 min. Dehydration was performed using a graded ethanol series of 60%, 70%, 80%, 90%, and 95% ethanol for 1 min each, 100% ethanol for 2 min, twice, and xylene for 5 min, 3 times. Images were captured using a BZ-710 All-in-One Fluorescence Microscope (KEYENCE Corporation, Osaka, Japan).

The frozen brain sections were rehydrated and treated with L.A.B. solution (Polyscience, Inc., Niles, IL, USA) for 20 min. They were then blocked with goat serum for 30 min, and incubated overnight at 4 °C with both the mouse-anti-Aβ (1:400) and rabbit-anti-NeuN (1:400, Cell Signaling Technology, Danvers, MA, USA) antibodies. Subsequently, sections were incubated in DyLight 594-labeled goat anti-mouse IgG and 488-labeled goat anti-rabbit IgG (1:400, Cell Signaling Technology, Danvers, MA, America) at room temperature for 1 h. Finally, the sections were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Institute of Biotechnology, Beijing, China) and sealed with anti-fluorescence quencher (Beyotime Institute of Biotechnology, Beijing, China). The sections were observed and photographed using a confocal fluorescence microscope (Leica, SP8., Wetzlar, Germany).

Paraffin-embedded P1 lower jaws (n = 5) were sliced to a thickness of 8 μm. Selected sections were then soaked in L.A.B. Solution (Polysciences, PA, USA) for 5 min for antigen activation, followed by incubation in 5% BSA/PBS for 1 h. After incubation with the primary antibodies for BMPRIA (sc-20736; Santa Cruz Biotechnology, CA, USA), BMPRIB (sc-25455; Santa Cruz Biotechnology), or BMPRII (MABD171; Merck Millipore, MA, USA), expression was detected using Alexa488- or Alexa594-conjugated secondary antibodies (Life Technologies). A fluorescence microscope (BZ-8000, KEYENCE Co, Osaka, Japan) was used for image analysis. Images were prepared using a BZ analyzer (KEYENCE) and Photoshop (Adobe Systems, CA, USA).