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Protocol
Rat and Mouse kidneys for LCM-proteomics
Keywords: Laser Capture Microdissection, LCM, hematoxylin, mouse, rat, kidney
Preparation of rat- and mouse kidneys suitable for Laser Capture Microdissection (LCM)-proteomics
The prtocol describes how to prepare rat- and mouse kidneys in a way suitable for Laser Capture Microdissection (LCM)-proteomics, on Hematoxylin stained sections.
The protocol have been used with both mouse (17 weeks old male C57Bl/6J strain) and rat (3 months old female Sprague Dawley) kidneys.
V1, October, 2009, Inserm U858, Joost Schanstra
Required Materials
- Rodents , C57Bl/6J, Sprague Dawley
- Pentobarbital , quantities based on weight, as indicated on the bottle
- PBS
- Liquid Nitrogen
- Filter paper , Whatman
- Eppendorf tubes , for kidneys from mice
- sterile 15ml centrifuge tubes , of Falcon-type, for kidneys from rat
- Freezer (-80)
Instructions
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Anaesthesia
Anaesthesia with pentobarbital. The quantities are determined by the weight of the animal and are described on the bottle/package.
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Perfusion
The animal should be immediately flushed with PBS. This is done by direct injection in the left ventricle of the heart by cutting (for pressure relief) the inferior vena cava (most accessible vein). Continue until the kidney “whitens".
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Dissection
Dissection and decapsulation of the kidneys.
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Freezing
The kidneys should be frozen on Whatman paper in N2(l) vapour until the kidney hardens (gets really white). This avoids the kidney “cracking” and losing kidney structure.
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Packaging
Insert the frozen kidney, with Whatman paper, in appropriate sized tube (1.5 ml for mouse and Eppendorf and 15 ml Falcon for rat) and transfer to the N2(l).
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Storage and transport
Stored for long-term storage at -80°C. Transport on dry-ice.
