Mass Purification Protocol for Drosophila melanogaster Wing Imaginal Discs: An Alternative to Dissection to Obtain Large Numbers of Disc Cells.

Drosophila melanogaster imaginal discs are larval internal structures that become the external organs of the adult. They have been used to study numerous developmental processes for more than fifty years. Dissecting these imaginal discs for collection is challenging, as the size of third-instar larvae organs is typically less than 1 mm. Certain experimental applications of the organs require many cells, which requires researchers to spend several hours dissecting them. This paper proposes an alternative to dissection in the form of a mass enrichment protocol. The protocol enables the recovery of many wing imaginal discs by grinding large quantities of third-instar larvae and separating the organs using filtration and a density gradient. The wing imaginal discs collected with this protocol in less than three hours are as well preserved as those collected by dissection. The dissociation and filtration of the extract allow the isolation of a large amount of wing imaginal disc cells.

Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of Critical Steps of Image Processing.

Apoptosis is associated with numerous phenotypical characteristics, and is thus studied with many tools. In this study, we compared two broadly used apoptotic assays: TUNEL and staining with an antibody targeting the activated form of an effector caspase. To compare them, we developed a protocol based on commonly used tools such as image filtering, z-projection, and thresholding. Even though it is commonly used in image-processing protocols, thresholding remains a recurring problem. Here, we analyzed the impact of processing parameters and readout choice on the accuracy of apoptotic signal quantification. Our results show that TUNEL is quite robust, even if image processing parameters may not always allow to detect subtle differences of the apoptotic rate. On the contrary, images from anti-cleaved caspase staining are more sensitive to handle and necessitate being processed more carefully. We then developed an open-source Fiji macro automatizing most steps of the image processing and quantification protocol. It is noteworthy that the field of application of this macro is wider than apoptosis and it can be used to treat and quantify other kind of images.