Inbedding van volwassen drosophilahoofden in paraffine: weefsel voorbereiden op histologie

- 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.