Mouse anti acetylated tubulin clone 6 11b 1

Staged animals were manually freed from chorions if necessary and fixed in 4 % formaldehyde overnight at 4 °C. The samples were washed in PBS and dehydrated through a methanol series. The embryos were stored in 100 % methanol at −20 °C until used.
All embryos were permeabilized in cold acetone at −20 °C and washed again in 100 % methanol. After rehydration by scalar washes in methanol dilutions, embryos were digested using Proteinase K (10 μg/ml) and subsequently fixed in 4 % formaldehyde for 20 min at room temperature. A blocking step with 10 % normal goat serum (NGS, Sigma-Aldrich) was carried out for 5–6 h at room temperature. Primary antibodies were added at a 1:1000 dilution in 10 % NGS overnight at 4 °C. The samples were washed in PBST (PBS, 0.1 % Tween20) and incubated with secondary antibodies (1:250 in 1 % NGS) overnight at 4 °C, protected from light. After several PBST washes, the samples were post-fixed and brought in glycerol through scalar washes.
The primary antibodies used in this study are: mouse anti-acetylated tubulin (clone 6-11B-1, T6793, Sigma-Aldrich), anti-Vasna, anti-Vasnb. Secondary antibodies used: goat anti-mouse Alexa647 (A21236, Molecular Probes), goat anti-rabbit Alexa488 (A11008, Molecular Probes).