Source: Sunderhaus, E. R., Kretzschmar, D. Mass Histology to Quantify Neurodegeneration in Drosophila .J. Vis. Exp. (2016).
This video describes how to embed Drosophila heads in paraffin using the collar method, a technique that prepares fly heads for serial sectioning. Paraffin sections are used for histological examinations of the brain's anatomy and for the assessment of neurodegeneration. The featured protocol demonstrates how multiple flies can be processed in one preparation, thereby increasing time-efficiency and minimizing experimental artifacts.
This protocol is an excerpt from Sunderhaus and Kretzschmar, Mass Histology to Quantify Neurodegeneration in Drosophila, J. Vis. Exp. (2016).
1. Fixing the Head on Collars and Embedding in Paraffin
NOTE: All of the steps in the fixation process should be done in a fume hood. Methylbenzoate, while not posing a health risk, has a highly distinct odor, which can be overwhelming if not handled in a fume hood.
Figure 1: Paraffin Serial Sections. A) Using the collar method, experimental and control flies can be processed as one sample by threading them onto one collar. Eyeless sine oculis flies are inserted for orientation (arrows). B) Schematic showing the orientation of the fly heads in the collar. C) In this image, sections from different fly heads are oriented left to right on the slide. From top to bottom on the slide, serial sections from the same fly head can be seen. In this case, a sine oculis fly was inserted at position three (arrow). The sections are stained by the fluorescent eye pigment that washes over the sections after cutting. Scale bar in A = 5 mm and in C = 0.5 mm. Please click here to view a larger version of this figure.
Collar | Genesee Scientific | TS 48-100 | We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. |
Rubber ice cube tray for embedding | Household store | The size can be made-to-fit by glueing in additional walls | |
Crystallizing dish | Fisher Scientific Company | 08-762-3 | |
Ether | Fisher Scientific Company | E138-1 | |
Ethanol | Decon Laboratories Inc. | 2701 | |
Choloroform | Fisher Scientific Company | C298-500 | |
Glacial Acetic Acid | Fisher Scientific Company | A38-212 | |
Methylbenzoate | Fisher Scientific Company | M205-500 | Distinct Odor – Use in fume hood! |
Low Melting Point Paraffin Wax | Fisher Scientific Company | T565 | Make sure to keep extra melted in a 65 °C waterbath |