Embedding Adult Drosophila Heads in Paraffin: Preparing Tissue for Histology
Trascrizione
– 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 a the rest of the body. For tissue fixation, place the collars di 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 a methyl benzoate, an organic solvent. Next, place it di a methyl benozoate and melted paraffin solution while heated a ensure the paraffin stays melted. Procedi a an elaborate series of washing steps with pre-heated paraffin alone a remove the methyl benzoate.
Next, rapidly place the collar di a mold and pour melted paraffin a cover it completely. Once the paraffin has hardened, remove the block from the mold and use a razor blade a trim excess paraffin. Break off the collar leaving the row of embedded heads di the paraffin block. In the example protocol, we will see the procedure demonstrated for the analysis of neurodegenerative phenotypes.
– To begin, first, use forceps a thread the subject flies by the neck into the collars. Align all the heads di the same orientation and ensure no damage occurs a the head or eyes. Include eyeless sine oculis flies at random known positions so that the order of the flies can be easily identified di the sections. Additionally, if the fly strain of interest has light or white eyes, thread red-eyed flies at intervals along the strip a ensure sufficient pigment is present a 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 a 4 hours. Perform a series of ethanol, methyl benzoate, and paraffin washes a 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 a 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 di the collar while the heads will stay di the paraffin block.
