Connexin 43 Immunohistochemistry in Larval Guts

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.