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Method Article
Immunostaining of spindle checkpoint proteins on Drosophila mitotic chromosomes from larval brains
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;
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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 However, the same method can be used for detecting also structural components of chromosomes (our unpublished results).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
Ethanol
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
Tweezers
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
Version History
CURRENT STATUS: Posted
Version 1
Posted 06 Feb, 2008
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Associated Publications
Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint
by Mariarosaria Musarò, Laura Ciapponi, Barbara Fasulo, +1
Nature Genetics (19 November, 2007)
Method Article
Immunostaining of spindle checkpoint proteins on Drosophila mitotic chromosomes from larval brains
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;
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 06 Feb, 2008
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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
Ethanol
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
Tweezers
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
Associated Publications
Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint
by Mariarosaria Musarò, Laura Ciapponi, Barbara Fasulo, +1
Nature Genetics (19 November, 2007)