Gt335

For Xenopus tropicalis, stage 46 tadpoles were fixed in 4% paraformaldehyde in PBS for 2 h at room temperature. The tadpoles were washed 3 × 15 min with PBS, and then the brains were dissected out. The brains were incubated for 1 h at room temperature in blocking buffer (3% BSA, 0.2% Triton X-100 in PBS), then incubated overnight at 4 °C in primary antibody against glutamylated tubulin (GT335, AdipoGen) diluted 1:1000 in blocking buffer [48 (link)]. Samples were then washed 3 × 15 min with PBS then incubated for 2 h at room temperature in secondary antibody (anti-mouse Alexa Fluor 488 or Alexa Fluor 594, Invitrogen) diluted 1:500 in blocking buffer, followed by 3 × 15 min washes with PBS. The brains were incubated for 1 h at room temperature in Alexa Fluor 647 phalloidin (Invitrogen) diluted 1:100 in blocking buffer, washed 3 × 15 min with PBS, then mounted between coverslips in ProLong Gold antifade reagent (Invitrogen). Immunostained brains were imaged on a Zeiss 710 confocal microscope.
For Xenopus laevis, stage 46 tadpoles were stained whole-mount according to Willsey et al. 2018 [49 (link)] using the primary antibody against glutamylated tubulin (GT335, AdipoGen) diluted 1:100. Following staining, brains were dissected and mounted in Vectashield (Vector Laboratories) and imaged on a Leica SP8 confocal microscope.

Primary antibodies used were against glutamylated tubulin (1:800 [tissue sections] and 1:500 [cell lines]; mouse; GT335; AdipoGen), pericentrin (1:250; rabbit; ab4448; Abcam), centrin (1:500; rabbit; N-17; Santa Cruz Biotechnology, Inc.), centrin (1:500; mouse; 20H5; EMD Millipore), A647-conjugated centrin (1:500; mouse; 20H5; EMD Millipore), γ-tubulin (1:500; mouse; GTU88; Sigma-Aldrich), α-tubulin (1:50; rat; YL1/2; AbD Serotec), p53 (1:100; mouse; DO-1; EMD Millipore), E-cadherin (1:30; rabbit; Cell Signaling Technology), and CP110 (1:250; rabbit; Jiang et al., 2012 (link)). The secondary antibodies FITC, Cy5, and rhodamine red (1:50 [tissue sections] and 1:200 [cell lines]; Jackson ImmunoResearch Laboratories, Inc.) as well as Alexa Fluor 488 and 647 (1:500 [cell lines]; Thermo Fisher Scientific) were also used.