Encyclopedia of Experiments: Biology
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- To immobilize flies for paraffin embedding, carefully push anesthetized Drosophila through the gap of a fly collar so their heads are uniformly positioned on top of the device while still attached to the rest of the body. For tissue fixation, place the collars in Carnoy's solution. Carnoy's is a denaturing fixation agent consisting of ethanol, chloroform, and acetic acid.
To prepare the sample for paraffin infiltration, dehydrate the tissue by transferring the collar through graded concentrations of ethanol. Then, transfer the collar to methyl benzoate, an organic solvent. Next, place it in a methyl benozoate and melted paraffin solution while heated to ensure the paraffin stays melted. Proceed to an elaborate series of washing steps with pre-heated paraffin alone to remove the methyl benzoate.
Next, rapidly place the collar in a mold and pour melted paraffin to cover it completely. Once the paraffin has hardened, remove the block from the mold and use a razor blade to trim excess paraffin. Break off the collar leaving the row of embedded heads in the paraffin block. In the example protocol, we will see the procedure demonstrated for the analysis of neurodegenerative phenotypes.
- To begin, first, use forceps to thread the subject flies by the neck into the collars. Align all the heads in the same orientation and ensure no damage occurs to the head or eyes. Include eyeless sine oculis flies at random known positions so that the order of the flies can be easily identified in the sections. Additionally, if the fly strain of interest has light or white eyes, thread red-eyed flies at intervals along the strip to ensure sufficient pigment is present to stain the slide.
Record the order of the flies together with the collar number if more than one is used. Once a collar is finished, place it into Carnoy's solution for 3.5 to 4 hours. Perform a series of ethanol, methyl benzoate, and paraffin washes to prepare the fly tissue for embedding. Next, place the collars into a rubber ice cube tray one per section.
Gently pour molten paraffin over the collars avoiding air bubbles and allow it to harden overnight. Remove the paraffin blocks containing the collars from the tray. Separate the paraffin block from the collar using a razor blade, gently breaking off the collar. The bodies will remain in the collar while the heads will stay in the paraffin block.
