Fluorophore conjugated anti igg antibodies

Samples were fixed in 4% (wt/vol) paraformaldehyde solution (Wako) for 15 min at room temperature on day 3 (for NVCM) and day 7 (for iPSC-CM) following seeding respectively. They were subsequently blocked and permeabilized with 2% (wt/vol) bovine serum albumin (BSA) (Gemini Bio-Products) solution with 0.1% (vol/vol) Triton X-100 (Promega) for 1 h at room temperature. Samples were then incubated with mouse monoclonal antibodies against alpha-actinin (1:100, Sigma) and rabbit monoclonal antibodies against connexin 43 (1:100, Cell Signaling Technology) overnight at 4 °C. Following primary antibody incubation, samples were washed three times with PBS at room temperature, followed by 1 h room temperature incubation with fluorophore-conjugated anti-IgG antibodies (1:200, Biotium). Hoescht 33258 (Invitrogen) nucleus counterstain was also performed. Following 3 times of washing with PBS (Gibco, Life Technologies), fluorescently stained samples were stored in PBS (Gibco, Life Technologies) with 0.05% (wt/vol) sodium azide (Alfa Aesar) at 4 °C and imaged within 1 week of staining.

All samples were fixed in 4% paraformaldehyde solution (PFA, Wako Chemicals) for 10 min at room temperature on day 3 following 3D printing. Fixed samples were then blocked and permeabilized using 2% bovine serum albumin (BSA) (Gemini Bio-Products) solution with 0.1% Triton X-100 (Promega) for 60 min at room temperature. Samples were subsequently incubated with mouse monoclonal antibodies against alpha-actinin (1:100, Abcam) and rabbit monoclonal antibodies against connexin 43 (1:100, Cell Signaling Technology) overnight at 4 °C. Following three washes with PBS at room temperature, samples were incubated with fluorophore-conjugated anti-IgG antibodies (1:200, Biotium). Fluorescently stained samples were stored in PBS with 0.05% sodium azide (Alfa Aesar) at 4 °C and imaged within 1 week of staining. Confocal microscope images were acquired with a Leica SP5 microscope (Leica Microsystems).

Samples were fixed in 4% (wt/vol) PFA solution (Wako) for 15 min at room temperature. Before imaging, fixed samples were blocked and permeabilized using 2% (wt/vol) bovine serum albumin (BSA) (Gemini Bio-Products) solution with 0.1% (vol/vol) Triton X-100 (Promega) for 1 h at room temperature. For human albumin and E-cadherin staining, samples were incubated with mouse monoclonal antibodies against human E-cadherin (1:100; Abcam) and rabbit monoclonal antibodies against human albumin (1:100; Abcam) overnight at 4°C. Following primary antibody incubation, samples were washed three times with D-PBS at room temperature. Secondary antibody incubation was carried out using fluorophore-conjugated anti-IgG antibodies (1:200, Biotium) in 2% (wt/wt) BSA (Gemini Bio-Products) solution for 1 h at room temperature. Hoechst 33342 (1:2000; Life Technologies) nucleus counterstain was also performed simultaneously with the secondary antibody incubation. Fluorescently stained samples were stored in D-PBS with 0.05% (wt/vol) sodium azide (Alfa Aesar) at 4°C after washing three times with D-PBS. Samples were imaged within one week of staining.