Connexin 43 antibody

For immunohistochemistry staining, dissected guts were fixed in 4% paraformaldehyde in PBS containing 0.01% Tween at 4°C overnight and then cryopreserved in 30% sucrose at 4°C. Guts were embedded in the OCT compound (Tissue-Tek, Sakura) and sectioned with 30µm thickness on a cryostat (Leica). Immunohistochemistry staining was performed as described previously (24 (link)). Briefly, to detect Connexin 43, sections were incubated with Connexin 43 Antibody (Cell Signaling, #3512) at a 1:500 dilution in blocking solution overnight at 4°C. After washing, a-conjugated Alexa 549 anti-rabbit antibody (1:500) was applied for overnight at 4°C. At least 8 sections in 6 larvae were analyzed in each condition and the experiment were repeated in at least three independent times.

The cells were fixed with a solution of 4% paraformaldehyde (VWR, Radnow, PA, USA) and 0.05% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 15 min. Each well was washed three times with PBS for 5 min after fixing. The cultures were then stained for nuclei (4,6-diamidino-2-phenylindole dihydrochloride, DAPI, ThermoFisher, Grand Island, NY, USA), actin (Alexa Fluor 488 Phalloidin, ThermoFisher, Grand Island, NY, USA), and sarcomeric α-actinin (Mouse Monoclonal Anti α-actinin; Sigma Aldrich, Inc., St. Louis, MO, USA). The wells were then washed with PBS three times to remove excess stain. Secondary staining was completed with goat anti-rabbit IgG secondary antibodies (Alexa Fluor 633, ThermoFisher, Grand Island, NY, USA). The wells were once more washed 3 times with PBS to remove excess staining. Afterwards, the wells were punched out with a commercially purchased metal square hole puncher and mounted onto glass microscope slides. ProLong Gold Antifade Mountant (ThermoFisher, Grand Island, NY, USA) was applied, and a microglass coverslip was placed on top to cover and seal the sample. Connexin 43 staining followed a similar protocol with just the addition of primary Connexin 43 Antibody (Cell Signaling, Danvers, MA, USA) and goat anti-Mouse IgG secondary antibody (Cyanine5, ThermoFisher, Grand Island, NY, USA). All staining was completed at a dilution of 1:1000 in PBS.