Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

This method can help answer key questions in the neuroscience field such as whether and how PLC activities in different brain regions are involved in brain function of the honeybee.The main advantage of this technique is that researchers can easily preform an experiment with the standard laboratory facilities as this protocol requires only basic manipulations.To begin, place a 50ml tube containing honeybees on ice for 30 minutes to anesthetize the bees.Next, prepare the dissection stage by folding a piece of dental wax in half and placing it in a plastic dish.Under a binocular microscope, use tweezers to separate the bee's head from its body.Then, fix the head on the dental wax and insert insect pins at the base of each antenna to hold the interior of the head in an upward position.Next, pour enough saline solution over the head to cover it.Using tweezers remove the antenna.After this, use a scalpel to make a horizontal cut near the bases of the antenna and a longitudinal cut at the border of the compound eyes.Make a horizontal cut at the top of the head and remove the cuticle to open a window over the brain.Next, use tweezers to remove the retin eye from the ossicle and the trache from the interior surface of the brain.Then, insert the tweezers directly under the eye cuticle to remove the connective tissue between the cuticle of the compound eyes and retin eye.After that, remove the cuticle and retin eye of the compound eyes.Next, use the tweezers to carefully remove the remaining trache from the interior surface of the brain.Then, cut the connective tissue between the brain and around the tissues and dissect the brain from the head capsule.Using the tweezers, peel the trache off of the posterior surface of the brain.Then use a scalpel to cut the connection between the mushroom bodies or MBs and other brain regions next to the vertical lobes.Place the dissected MBs and any remaining brain tissues into 1.5ml tubes and freeze them rapidly with dry ice.First, add 10ml of homogenization buffer to the frozen brain tissue.Use a plastic peselle to manually homogenize the tissue in the 1.5ml tube.After this, transfer the homogenized tissue solution to the next tissue in the lot and repeat the homogenization process.Then, add 30