Chromosome spreads were performed as previously described (Wahba et al., 2013 (link)). Slides were incubated with a mouse anti-FLAG monoclonal (Sigma-Aldrich) at 1:2000, mouse anti-V5 (Life Technologies, Carlsbad, CA) at 1:2000, polyclonal rabbit anti-Pds5 at 1:2000, or polyclonal rabbit anti-Mcd1 at 1:2000 dilutions. The primary antibody was diluted in blocking buffer (5% bovine serum albumin, 0.2% milk, 1× phosphate-buffered saline, and 0.2% Triton X-100). The secondary Cy3-conjugated goat anti-mouse antibody (115-165-003) was obtained from Jackson ImmunoResearch (West Grove, PA) and diluted 1:2000 in blocking buffer. Indirect immunofluorescence was observed using an Olympus IX-70 microscope with a 100×/NA 1.4 objective and Orca II camera (Hamamatsu, Bridgewater, NJ).
Protocol detail
Immunofluorescence Microscopy of Chromosome Spreads
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Anti antibody
Antibody
Bovine serum albumin
Buffer
Chromosome
Dilutions
Goat
Indirect immunofluorescence
Microscope
Milk
Mouse
Orca
Phosphate
Rabbit
Saline
Triton x 100
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Variable analysis
independent variables
- Antibody type (mouse anti-FLAG monoclonal, mouse anti-V5, polyclonal rabbit anti-Pds5, polyclonal rabbit anti-Mcd1)
- Indirect immunofluorescence (observed using an Olympus IX-70 microscope)
- Dilution of primary antibodies (1:2000)
- Dilution of secondary Cy3-conjugated goat anti-mouse antibody (1:2000)
- Blocking buffer composition (5% bovine serum albumin, 0.2% milk, 1× phosphate-buffered saline, and 0.2% Triton X-100)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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