Immunostaining of spindle checkpoint proteins on Drosophila mitotic chromosomes from larval brains

doi:10.1038/nprot.2008.25

Method Article

Immunostaining of spindle checkpoint proteins on Drosophila mitotic chromosomes from larval brains

https://doi.org/10.1038/nprot.2008.25

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Biochemistry

Computational biology and bioinformatics

Biological techniques

chromosome cytology

immunostaining

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 However, the same method can be used for detecting also structural components of chromosomes (our unpublished results).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

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Immunostaining of spindle checkpoint proteins on Drosophila mitotic chromosomes from larval brains

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