Jacobs:Tubulin Immunostaining Protocol

Immunostaining Protocol			FH / MED April 2010

This is a protocol developed for staining alpha tubulin (Abcam, ab52866) and gamma tubulin (Sigma T6557) in IMCD cells, based on Attias 2010, doi:10.1159/000262317. It can be used as a reference, but depending on your antibodies you might consider other fixation protocols such as ice cold methanol or acetone. Dilutions of antibodies have to be determined by titration. When using multiple primaries at the same time, primary’s host must be of different species. biocompare.com is a useful resource of commercially available antibodies Example: primary: rabbit anti alpha tubulin IgG, secondary donkey anti rabbit IgG Alexa 555 Materials:
 * Circular cover glass 12 mm 1.5
 * Triton X-100
 * Paraformaldehyde (PFA)
 * PBS
 * Bovine Serum Albumin
 * Prolong Anti Fade mounting medium
 * Primary and secondary antibodies

Preparation:
 * 1) 	Plate cells on 12mm circular 1.5 glass coverslips the day before in 24 well plate (50’000 cells/well)
 * 2) 	Prepare 0.5% Triton X-100 in PBS (Triton X is very viscous, so prepare a few days before and make serial dilutions)
 * 3) 	Prepare 4% Paraformaldehyde in PBS
 * 4) 	Perform all handlings of Paraformaldehyde inside fume hood and wear gloves (PFA is toxic!)
 * 5) 	Heat PFA in PBS to 60°C on heating plate and use magnetic stir bar to mix until PFA dissolves and solution becomes clear
 * 6) 	Prepare 3% Bovine Serum Albumin (BSA) in PBS

Staining:
 * 1) 	Wash cells twice with pre-warmed PBS
 * 2) 	Fix cells with 500 µL 4% PFA in PBS for 15 min at RT in fume hood
 * 3) 	Aspirate and Wash 3x with PBS (discard all washes into toxic waste container)
 * 4) 	Permeabilize cells with 500 µL 0.5% Triton-X in PBS for 5 min
 * 5) 	Aspirate and wash 3x with PBS
 * 6) 	Block with 3% BSA in PBS for 30 min at RT
 * 7) 	Prepare primary antibodies (dilute in 0.5 % BSA in PBS)
 * 8) 	Aspirate blocking solution, add primary antibodies
 * 9) 	Allow to sit overnight at 4°C (decreases background), or 1-2 hours at room temperature
 * 10) 	Wash 3x with PBS (Allow 20 min between washes)
 * 11) 	Add secondary antibodies (dilute in 0.5% BSA in PBS)
 * 12) 	Wash 3x with PBS (Allow 20 min between washes)
 * 13) 	Mount with ProlongAntiFade mounting medium, let it cure for 24 hours at room temperature protected from light
 * 14) 	Seal the coverslips with nailpolish