Rat anti tyrosinated tubulin

Drosophila pupal wings were prepared for imaging as previously described (Axelrod, 2001 (link)). Primary antibodies were as follows: mouse anti-Fmi (#74, Developmental Studies Hybridoma Bank), mouse anti-Arm (Developmental Studies Hybridoma Bank), rabbit anti-alpha Tubulin (Abcam, Cambridge, UK), rat anti-tyrosinated Tubulin (Abcam, Cambridge, UK), rat anti-Ds and rat anti-Ft (Yang et al., 2002 (link)). Images were acquired on a Leica TCS SP5 AOBS confocal microscope using a 63x objective and processed with LAS AF (Leica).

Primary antibodies used were chick anti-GFP (1:1000; Abcam), rabbit anti-TDP43 (1:1000; Cell Signalling), rabbit anti-neurofilament-H (1:1000; Millipore), rat anti-tyrosinated tubulin (1:1000; Abcam) and rabbit anti-Glu-tubulin (1:1000; Abcam). Secondary antibodies were anti-rabbit Cy-3-conjugated, anti-chick Alexafluor488, anti-rabbit Alexafluor 568 and anti-rat Alexafluor 568 (all at 1:1000; Life Technologies).
Coverslips were fixed by adding 400 µl of 4% paraformaldehyde (PFA)/15% sucrose (Sigma-Aldrich) to each well for 15 mins at room temp, then washed twice with PBS-T (1 X phosphate-buffered saline (PBS) + 0.1% Triton X-100) and rocking. Non-specific binding was blocked using 0.5% bovine serum albumin (BSA; Sigma) in PBS for 1 h at room temp. Coverslips were incubated in primary antibodies diluted as above in 0.1% PBS-T with 0.5% FBS, overnight on a rocker at 4°C, then washed 3 X 10 mins with PBS-T on a rocker plate at room temperature, followed by incubation in secondary antibodies as above in PBS-T for 1–2 h at room temperature with rocking. After further washes, the coverslips were mounted on slides using Fluorsave (Calbiochem) with Hoechst reagent (Thermo-Fisher Scientific).

Mouse anti-βII-spectrin was from BD Biosciences (US, cat. #612563) and used 1:100. Rat anti-tyrosinated tubulin was from Abcam (cat. #ab6160) and used 1:200. Mouse anti-alpha tubulin was from Sigma-Aldrich (clone α-3A1, cat. #T5168) and used 1:400. The corresponding secondary antibodies for widefield were Alexa Fluor 488 and 568 (Thermo Fisher Scientific, anti-mouse-AF488 cat. # A32723, anti-rat-AF568 cat. #A11077); and anti-mouse ATTO-647N was used for STED (Sigma-Aldrich, cat. #50185).