Immunofluorescence Staining for Acetylated and Detyrosinated Tubulin

Cells were grown on glass coverslips and then fixed in 100% cold methanol (10 min, 4 °C). Immunofluorescence staining was performed as previously described [10 (link)] with the following antibodies: Anti-acetyl-α-tubulin (Lys40) (D20G3) XP Rabbit mAb (1:1000; Cell Signaling), Anti-detyrosinated-α-tubulin (1:1000; Abcam), Monoclonal Anti-detyrosinated- α-tubulin (1:10,000: RevMAb), Monoclonal Anti-α-tubulin Clone DM1A (1:5000; Sigma Aldrich) and β-actin (1:2000; AC-15, Sigma). Anti-IgG antibodies conjugated to Alexa-488 (1:1000; Molecular Probes) were used for secondary detection and Hoescht 33342 (1:5000; Sigma) was used for nuclear staining. Images were acquired using an Olympus FV1000 laser scanning confocal microscope (Olympus, Center Valley, PA). The deTyr-Tubulin antibody (RevMab) was developed by Takashi Hotta and Ryoma Ohi [23 (link)] and is now commercially available through RevMab under catalogue number RM444 (https://www.revmab.com/index.php/product/anti-detyrosinated-alpha-tubulin-rabbit-monoclonal-antibody-clone-rm444/).