Immunohistochemistry (IHC) helps identify different types of tissues and provides important information about the pattern, shape, and structure of cells in a tissue sample. It is a very useful tool and widely used to uncover histology and pathology diagnosis uses. The IHC is a very useful tool to conduct research in the developmental biology area. Some insects, like butterflies, bees, beetles, ants, wasps, and moths, undergo a unique life cycle called complete metamorphosis, which has four distinct stages: egg, larva, pupa, and adult. Larvae are often wingless and have different habits and forms than adults, which are better suited for growth and development. During the metamorphosis, how does the insect legs change is a really an interesting issue.To better understand insect development, agriculture pest control, and insect pathogen interaction, here we use an insect Bombyx mori (domestic silk moth) as a model to detail how to conduct the IHC assays, such as HE staining, TUNEL to see the cell apoptosis, pH3 and BrdU staining to check cell division. The reagents can be easily found, and this step-by-step protocol is easily reproduced.

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 filters, zprojection and thresholding. Even though it is commonly used in imageprocessing 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 can allow or not to detect subtle differences of the apoptotic rate. On the contrary, images from anticleaved caspase staining are more sensitive to handle and proved to necessitate to be 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 as it can perfectly be used to treat and quantify other kind of images.