Materials and reagents

Reagents

Immunofluorescence labeling

1. Primary antibodies against the protein(s) of interest (the Mad1 example is shown here):

   1. Mad1 (Thermo Fisher Scientific, catalog number: PA5–28185). Other alternatives compatible with immunofluorescence staining may be used after empirical optimization.

   2. CREST (to stain kinetochores) (Antibodies Incorporated, catalog number: 15–234). Other alternatives staining the kinetochore or the centromere that are compatible with immunofluorescence staining may be used after empirical optimization for specificity.

   3. α-Tubulin, DM1 alpha (Sigma, catalog number: T9026)

2. Secondary antibodies tagged with fluorophore:

   1. Alexa Fluor^® 488 AffiniPure Donkey Anti-Rabbit IgG (Jackson ImmunoResearch Inc., catalog number: 711–545-152)

   2. Alexa Fluor^® 594 AffiniPure donkey Anti-Mouse IgG (Jackson ImmunoResearch Inc., catalog number: 715–585-150)

   3. Cy^TM 5 AffiniPure Donkey Anti-Human IgG (Jackson ImmunoResearch Inc., catalog number: 709–175-149)

      Note: Other alternative fluorophore-conjugated secondary antibodies compatible with immunofluorescence staining may be used after empirical optimization.

3. Sodium chloride (NaCl) (SRL, catalog number: 64072); may be procured commercially from other standard sources

4. Potassium chloride (KCl) (SRL, catalog number: 38630); may be procured commercially from other standard sources

5. Disodium hydrogen phosphate (Na₂HPO₄) (SRL, catalog number: 1949144); may be procured commercially from other standard sources

6. Potassium dihydrogen phosphate (KH₂PO₄) (SRL, catalog number: 1649201); may be procured commercially from other standard sources

7. Triton X-100 (Sigma, catalog number: T8787–100ML)

8. Bovine serum albumin (BSA) (pH 6–7) (SRL, catalog number 9048–46-8); may be procured commercially from other standard sources

9. 4',6-diamidino-2-phenylindole (DAPI), 5 mg/mL stock solution made in dimethyl sulfoxide (DMSO) (Thermo Scientific, catalog number: 62247)

   Note: Alternatively, chromatin dyes for immunofluorescence such as Hoechst 33342 may be used after empirical optimization.

10. ProLong Diamond^TM mounting media (Thermo Scientific, catalog number: P36970). Alternative antifade mounting media with a refractive index compatible with the lens and the lens immersion medium may be used after empirical optimization

    
    

1. 1× phosphate buffered saline (PBS) (see Recipes)

2. Blocking solution (see Recipes)

   
   

1. 1× phosphate buffered saline (PBS)

   Components	Amount/volume added	Working concentration
   NaCl	8 g	137 mM
   KCl	0.2 g	2.7 mM
   Na2HPO4	1.44 g	10 mM
   KH2PO4	0.24 g	2 mM
   Deionized water	Make up the volume to 1 L

   
   

2. Blocking solution for immunofluorescence staining

   Components	Amount/volume added	Working concentration
   PBS	50 mL	1×
   Triton X-100	250 µL	0.5%
   BSA	0.5 g	1%

   
   

   1. Weigh 0.5 g of BSA and add to 45 mL of 1× PBS in a 50 mL tube.

   2. Place the tube on a rocker and mix at slow speed until completely dissolved. Avoid frothing of the solution.

      Caution: High-speed mixing can denature proteins including BSA, of which frothing is a sign.

   3. Add 250 μL of Triton X-100 to the solution.

   4. Slowly mix the solution on a rocker to dissolve all the components completely.

   5. Add 1× PBS to make up the volume to 50 mL and gently mix to homogeneity.

      
      
   Laboratory supplies

   1. Clean glass slides and coverslips

      Critical: The coverslips should be properly cleaned and ideally be of category #1.5 (even thickness between 0.16 and 0.19 mm) to minimize chromatic aberration and refractive index mismatches between the lens, immersion medium, and sample mounting medium.

   Equipment

   Confocal image acquisition

   1. Leica TCS SP8 laser scanning optical confocal microscope with HCX PL APO 63X-1.4 NA oil-immersion objective and a HyD (hybrid) or photomultiplier tube (PMT) detector.

      Note: Equivalent confocal microscopes from standard commercial sources may be used, as long as they offer the following key features:

      1. Plan apochromat (PL APO) confocal grade objectives with a high numerical aperture (1.2 or above).

      2. Laser illumination in at least three distinct colors.

      3. Emission pinhole(s) designed to block out-of-focus emission light from adjacent z-planes from being captured in the images.

      4. In the case of raster laser scanning confocal scan-heads, high-sensitivity detectors such as PMTs, hybrid detectors, or avalanche photodiodes (APDs) with a quantum efficiency in the visible spectrum around 50 % may be used. Alternatively, for spinning disc confocal microscopes, high sensitivity cameras such as scientific complementary metal oxide semiconductor (sCMOS) cameras or electron multiplying charge-coupled device (EMCCD) cameras, with a quantum efficiency in the visible spectrum of around 80% or above, may be used.

   Software and datasets

   For image analysis

   1. IMARIS software suite, version 8.3, Bitplane. Higher versions may be used.

   2. Fiji (open-source image analysis software) (https://fiji.sc, Schindelin et al. [9])

      
      

   For statistical analysis

   1. GraphPad Prism (GraphPad Software, USA). Other standard software packages may be used.

   Procedure