Alexa fluor 488 goat anti mouse

The Nr-CWS was provided by Liaoning Greatest Bio-Pharmaceutical Co., Ltd. The high glucose medium, fetal bovine serum, and 0.25% EDTA trypsin were purchased from Gibco (San Jose, CA, USA). The streptomycin-penicillin was purchased from Thermo (Waltham, MA, USA). The PBS, CCK-8 kit and RNA rapid extraction kit were purchased from Yishan (Shanghai, China). The 0.8 μM transwell chambers, DMSO, DAPI, Alexa Fluor 488 Goat anti-mouse, β-actin antibody, and GAPDH antibody were purchased from Sigma (St. Louis, MO, USA). The FITC-Dextran (MW4000) was purchased from MCE (Princeton, NJ, USA). The TNF-α and TGF-β ELISA kits were purchased from Lianke Bio (Hangzhou, China). The Arg-1 and iNOS antibodies were purchased from Bio Legend (San Diego, CA, USA). The H&E staining kit, quantitative PCR kit, and reverse transcription kit were purchased from Yeason (Shanghai, China). The Masson staining Kit was purchased from Sbjbio (Nanjing, China). The 4% paraformaldehyde, anti-fluorescence quenching blocking solution, and immunohistochemical blocking solution were purchased from Beyotime (Shanghai, China). The primers were purchased from Sangon Biotech (Shanghai, China). The mTOR/p-Mtor, Akt/p-Akt, PI3K/p-PI3K, and TGF-β antibodies were purchased from Abcam (Cambridge, UK).

We fixed the brain organoids at 8 weeks of differentiation with 2% paraformaldehyde for 45 min, blocked with 10% goat serum, 1% bovine serum albumin (BSA), and 0.15% saponin in 1× PBS for 1 hour and stained with primary antibodies diluted in blocking solution for 48 hours. After three washes with 1% BSA/0.15% saponin in 1× PBS, we stained the organoids with secondary antibodies for 24 hours, stained them with Hoechst for 1 hour, washed twice, and mounted them on the glass slide with Immu-Mount. We used the following antibodies: mouse anti-Map2 (clone AP-20, Sigma-Aldrich), mouse anti–β-III-tubulin (clone SDL.3D10, Sigma-Aldrich), rabbit anti-GFAP (polyclonal, Dako), rabbit anti-Nestin (polyclonal, Sigma-Aldrich), Alexa-Fluor 488 goat anti-mouse, and Alexa-Flour 568 goat anti-rabbit immunoglobulin G.

MC3T3-E1 mouse osteoblast cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco 1X) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin and 1% GlutaMAX. The cells were kept in a 5% CO₂ incubator at 37 °C and the medium was changed every 2–3 days. To stain the cells for fluorescent imaging, MC3T3-E1 cells on PDMS platforms were washed with 1% phosphate buffered saline (PBS) twice and fixed with 4% paraformaldehyde for 15 min at room temperature. Throughout the staining process, PBS was used to wash the cells in between each step. Cells were permeabilized in 0.1% Triton X-100 solution for 12 min before immersed in blocking solution with 1% bovine serum albumin for 30 min. Cells were then incubated with the primary antibody mouse anti-vinculin (Sigma-Aldrich) over night before being washed and incubated with secondary antibody Alexa Fluor 488 goat anti-mouse (Sigma-Aldrich) for 2 h. Following this, 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) was used as a counterstain to stain the cell nuclei for 30 min. After washing the cells in Alexa Fluor 555 phalloidin (Sigma-Aldrich) for 2 h to stain F-actin, the cells were subsequently washed with PBS thrice in preparation for imaging using a Nikon upright microscope.