Authors: Mariarosaria Musar
We describe a very simple method to immunostain Drosophila mitotic chromosomes for spindle checkpoint proteins and kinetochore components. By using appropriate primary antibodies, this procedure allowed us to detect Zw10, Zwilch, Cenp-C, Cenp-meta and BubR1 on kinetochores of larval brain chromosomes. However, the same method can be used for detecting also structural components of chromosomes (our unpublished results).
- Saline (0.7% NaCl),
- Hypotonic solution (0.5% sodium citrate).
- Fixing solution (1.8% Formaldehyde, 45% acetic acid in dH20)
- Liquid nitrogen
- PBS-T (PBS containing 0.1% TritonX).
- Appropriate primary and secondary antibodies
- Vectashield medium H-1200 with DAPI (4,6 diamidino-2-phenylindole)
- Slides and coverslips
- Humid box
- Dissecting Scope
- Epifluorescence Microscope
- Dissect Drosophila larval brains in saline (0.7% NaCl),
- Treat dissected brains for 10 min with a hypotonic solution of 0.5% sodium citrate.
- Fix for 5 min in a drop of fixing solution (1.8% Formaldehyde, 45% acetic acid) on a coverslip.
- Gently lean a clean slide over the coverslip and squash the brain in the same fixing solution
- Freeze the slide in liquid nitrogen
- Remove the coverslip and immerse the slide in cold ethanol (-20°C) for 10 minutes.
- Wash the slide in PBS-T (PBS containing 0.1% TritonX).
- Incubate the slide overnight with appropriate primary antibody in a humid box at 4°C
- The next day, wash the slide twice in PBS-T for 10 minutes
- Incubate the slide with the secondary antibody for 2 h at room temperature, in a humid box.
- Wash the slide twice in PBS-T for 10 minutes and let it air dry.
- Mount the slide in Vectashield medium H-1200 with DAPI (4,6 diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA) to stain DNA and reduce fluorescence fading.
- Analyze the immunostaining with a epifluorescence microscope
Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint, Mariarosaria Musarò, Laura Ciapponi, Barbara Fasulo, Maurizio Gatti, and Giovanni Cenci, Nature Genetics 40 (3) 362 - 366 03/02/2008 doi:10.1038/ng.2007.64
Mariarosaria Musar, DiSTeBA, Universit del Salento, Lecce, 73100 Italy; Istituto di Biologia e Patologia Molecolari del CNR and Dipartimento di Genetica e Biologia Molecolare, Universit di Roma La Sapienza, Roma, 00185 Italy
Correspondence to: Mariarosaria Musar ([email protected])
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.25. Originally published online 6 February 2008.