Goat anti rabbit igg biotinylated secondary antibody

Cerebral organoids were fixed with 3.75% acrolein and 2% paraformaldehyde in 0.1M phosphate buffer (PB; pH7.4) overnight at 4°C(Milner et al., 2011 (link)). The next day, the sections were rinsed in PB. Two experiments were performed. Experiment 1: Normal Morphology-Following en bloc staining with uranyl acetate and graded ethanol dehydration, samples were embedded in an Epon analog resin. Ultrathin sections (65 nm) were contrasted with lead citrate for use in electron microscopy. Experiment 2: Connexin-43 Immunolabeling – Free-floating cerebral organoids were incubated with anti-connexin-43 (Sigma; 1:2000) and a goat-anti-rabbit IgG-biotinylated secondary antibody (Jackson Immunoresearch Laboratories; 1:400) using the avidin-biotin complex peroxidase method (Vectastain ABC-HRP Kit; Vector Laboratories) (Milner et al., 2011 (link)). Organoids were dehydrated and flat-embedded in EMBed-812. Organoids were sectioned (70 nm thick) on a Leica ultratome (Ultracut UCT) and collected on 400 mesh copper grids and then counterstained with uranyl acetate and Reynold’s lead citrate. For both experiments, grids were imaged on an FEI Tecnai BioTwin Transmission Electron Microscope. Elements were identified using morphological criteria defined in Peters et al. (1991) . The number of organoids used per experiment is stated for each individual figure.

Muscle sections were fixed in 4% PFA for 10 minutes, washed in PBS, then incubated in 0.5% Triton X-100 diluted in PBS for 10 minutes. Sections were rinsed in PBS and incubated with 3% H₂O₂ for 10 minutes to block endogenous peroxidases. After being washed with PBS, muscle sections underwent heat-mediated antigen retrieval in sodium citrate (10 mM, pH 6.5) for 20 minutes at 92 °C. Sections were then washed in PBS and block in 2.5% NHS for 90 minutes. Following blocking, sections were incubated in primary antibody overnight for Rb IgG anti-HMGB1 (1:250, ab18256; Abcam) and mouse IgG2b anti-dystrophin (1:200, D8168 Sigma; Millipore Sigma) diluted in NHS. The next day, sections were washed with PBS, incubated for 75 minutes in goat anti-rabbit IgG biotinylated secondary antibody (1:1000; Jackson ImmunoResearch) diluted in NHS, washed in PBS, and then incubated for 75 minutes in streptavidin-conjugated AF594 (1:250; Invitrogen) and goat anti-mouse IgG2b AF488 (1:250, A21141; Invitrogen) diluted in PBS for 75 minutes. Sections were then rinsed in PBS, stained with DAPI (1:10,000; Invitrogen) for 10 minutes and mounted with VectaShield fluorescent mounting media (Vector Labs).