Monoclonal rabbit anti acetyl α tubulin antibody

Chlamydomonas cultures grown overnight in liquid TAP media (200 μL) were allowed to adhere for ten minutes onto glass slides pre-treated with 0.1% poly-L-lysine (Sigma). Slides were then incubated twice with methanol at −20°C for 4 minutes, excess methanol was removed and slides were dried. Following blocking for 1 hour at RT with 10% goat serum (Sigma) in PBS, samples were incubated for 3–4 hours at RT with a monoclonal mouse anti-HA.11 antibody (BioLegend) diluted 1:200 and with a monoclonal rabbit anti-acetyl-α-tubulin antibody (Cell Signaling Technology) diluted 1:500. Slides were washed trice with PBS and incubated with secondary antibodies diluted 1:1000 for 1 hour at RT. Secondary antibodies used were Alexa Fluor 647 goat anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen). Slides were washed three times with PBS and Prolong Gold Antifade Reagent (Cell Signaling Technologies) was added to maintain samples until imaging. Images were acquired on a confocal Nikon Ti-Eclipse inverted microscope with a Nikon Plan Apo 100 × 1.40 oil immersion objective and an Andor iXon EM-CCD camera using μManager software package⁷⁰ .