Comprehensive Immunostaining Techniques

Transmission electron microscopy was performed as previously described (36 (link)). Hematoxylin & eosin staining was carried out as described (37 (link)). For immunostaining, embryos or tissues were fixed with 4% paraformaldehyde overnight at 4°C and then rinsed with PBS 3 times. The samples were dehydrated in an increasing gradient of 10% to 30% sucrose/PBS and embedded in OCT (Tissue Tek). The samples were cryosectioned at 10-μm thickness, and OCT was washed out by PBS. The sections were incubated with diluted primary antibody overnight at 4°C and then with cy2- or cy3-labeled secondary antibody (Jackson Immunoresearch) for 1 hour at room temperature with PBS wash 3 times in between. The following antibodies were used for immunostaining: rabbit-anti-Numb (Cell Signaling, 2756s, 1:200 dilution), mouse anti-α-Actinin (Sigma, A7811, 1:500 dilution), mouse anti-α-Actin (Sigma, A9357, 1:500 dilution), goat anti-NEBL (Abcam,ab99420, 1:100 dilution), mouse anti-α-Tubulin (Covance, MMS-407R, 1:800 dilution), mouse anti-Desmin (Covance, MMS-454s, 1:200 dilution), and Phalloidin (Invitrogen, A12379 1:500 dilution).