Spindle Angle Measurements

Figure 1. Spindle angle measurement principle. Spindle poles coordinates are measured, then the spindle angle alpha is calculated.

Materials and Reagents

1. Fixed cells
   
2. Antibodies
   For example, for mitotic spindle poles, an antibody against γ-tubulin antibody (clone AK-15) (e.g. Sigma-Aldrich, catalog number: T3320 )
   For mitotic spindle using an antibody directed against α-or β-tubulin (clone TUB 2.1) (e.g. Sigma-Aldrich, catalog number: T4026 )
   Anti-rabbit coupled to Alexa Fluor^® 555 (Life Technologies, Invitrogen™, catalog number: A21429 )
   Anti-mouse coupled to Alexa Fluor^® 488 (Life Technologies, Invitrogen™, catalog number: A11029 )
   Note: Primary (AK-15 and TUB 2.1) and secondary (Anti-rabbit coupled to Alexa Fluor^® 555 and anti-mouse coupled to Alexa Fluor^® 488) antibodies were used after a 1,000 time dilution in 1x PBS/1% BSA.
   
3. 10x PBS (Life Technologies, Invitrogen™, catalog number: AM9625 )
   
4. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, catalog number: D9542 )
   
5. BSA (Sigma-Aldrich, catalog number: A4503 )
   
6. 1x PBS (see Recipes)
   
7. 1x PBS/1% BSA (see Recipes)
   

Equipment

1. We use a Zeiss Axioimager Z1 with 63x Plan-Apochromat 1.4 oil lens and using an Axiocam Mrm camera with a Grid Projection Illumination (apotome). The system is driven by Axiovision software. Images can also be obtained from any confocal microscopes/widefield microscope + deconvolution.
   
2. 12 mm round coverslips (#1.5)
   
3. Glass slides
   
4. Vectashield^® Mounting Media (Vector Laboratories, catalog number: H-1000 ) or ProLong^® Gold (Life Technologies, Invitrogen™, catalog number: P36934 )
   

Software

1. Axiovision software
   
2. Open source software ImageJ 1.47q (http://rsbweb.nih.gov/ij/index.html)
   
3. Macro for spindle angle measurements
   

Procedure