Mouse anti tyrosinated tubulin

After washing in PBS, sections were treated with PBS—0.1%, Triton X-100—10% serum for 30 min. They were then incubated overnight at 4 °C with the following primary antibodies diluted in the blocking buffer: mouse anti-acetylated tubulin 1:100 (Sigma); mouse anti-α-tubulin (1:100, Sigma-Aldrich); rabbit anti-Cenpj: 1:200 (Proteintech); rabbit anti-Cdk5rap2 (1:100, Milipore); rabbit anti-centrin: 1:100 (Milipore); chicken anti-GFP (1:700, Millipore); rabbit anti-pH3 (1:200, Upstate); mouse anti-γ-tubulin (1:100, Abcam) and mouse anti-tyrosinated tubulin 1:100 (Sigma). Sections were then incubated with appropriate fluorescent secondary antibodies. To detect Cenpj, a step of antigen retrieval with citrate was performed before the blocking (sodium citrate pH=6 for 20 min at 90 °C).

Fixed hatching and first lorica larvae were washed three times for 5 min in PTw to remove sodium azide, and perforated afterwards with a thin needle to allow antibody penetration through the larval cuticle. Perforated larvae were transferred into PBS with 0.5% Triton X-100 (PTx) for permeabilization for 2 h at room temperature, and subsequently blocked in 1% bovine serum albumin (BSA) in PTx for 2 h at room temperature. Before adding the primary antibody, larvae were blocked in 10% normal goat serum (NGS) in PTx twice for half an hour. The analysed primary antibodies (mouse anti-acetylated tubulin (Sigma, #T6793), mouse anti-tyrosinated tubulin (Sigma, #T9028), rabbit anti-pCaMKII (Santa Cruz Biotechnology, #sc-12886), rabbit anti-serotonin (Sigma, #S5545) and rabbit anti-FMRFamide (Immunostar, #20091)) were diluted 1 : 100 in 10% NGS in PTx and incubated for approximately 40 h at 4°C. Continuous washes in 1% BSA in PTx for approximately 7 h to remove the primary antibody were followed by blocking in 10% NGS in PTx twice for half an hour and incubation in Alexa-conjugated secondary antibody diluted 1 : 250 in 10% NGS in PTx for approximately 40 h at 4°C. Finally, secondary antibodies were washed out in PTx, and if needed nuclei were counterstained with Sytox Green.