The most common reason for generating antibodies against a particular protein in Drosophila is to examine its pattern of endogenous expression during development. There are many methods that use the exquisite sensitivity of antibodies to locate a cellular component or a specific molecule (a tissue antigen). This process of localizing proteins in cells of a tissue section exploits the principle of antibodies binding specifically to antigens in biological tissues. The interaction between antibody – antigen can be visualised in different ways like an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction or the antibody can also be tagged to a fluorophore, such as FITC, rhodamine, Texas Red.
The antibodies used for specific detection can be polyclonal or monoclonal. Monoclonal antibodies are generally considered to exhibit greater specificity. Polyclonal antibodies are made by injecting animals with peptide antigens, and then after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes. Antibodies can be classified into primary and secondary ,where Primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabelled), while secondary antibodies are raised against primary antibodies.
Most of the immunohistochemical methods used today are a modification of protocols first developed by Zalokar and Erk ( 1977 ) and Mitchison and Sedat ( 1983 ). Subsequently many modifications were done ,which is mostly due to improvement in antibody reagents and Microscopy techniques. As mentioned above immunostaining / Antibody staining offers and excellent means to locate the distribution of your cloned gene in Drosophila embryo or larvae , which involves several steps like fixing, staining ,dissecting and mounting. The standard protocol can be found on many Drosophila lab websites , but the main purpose of this post is to understand the importance of the components used during the steps of fixing, processing and staining.
Take any protocol all includes the steps of fixtaion and processing before they can be stained for the antibodies. Drosophila embryos are protected by an outer chorion layer, as well as an impermeable and opaque vitelline membrane. So first step after collection of embryos includes removal of chorion layer and this done for 50% bleach ( sodium hypochlorite) for about 3 minutes.
Fixation can be performed by equal mix of fixative ( includes 4 – 10 % formaldehyde or para formaldehyde depending on the tissue or protocol used and PBS 1X ) and Heptane. Two layers are formed when these two solutions are mixed – Fixative at the bottom and heptane forms the upper layer. So when dechorinated embryos are added to this mix , the embryos sink in heptane and settle at the interface of two layers . The heptane will then become saturated by fixative , allowing the fixative to enter the hydrophobic vitelline membrane surrounding the embryos.
After fixed for about 20 minutes , the lower layer of fixative will be removed leaving heptane in the eppendorf tube . To this add methonol and shake vigorously for about 1 minute. This will break the vitelline membrane and embryos will settle at the bottom of the tube. The settled embryos can be used for staining or can be stored in absolute ethanol.
When it comes to staining, two important components are detergent and BSA . Detergents like Tween-20 or Triton-X will permeabilize the cell membrane for uptake of antibodies and BSA ( Bovine serum albumin) will help in blocking non specific antibody binding sites.
Image gallery of Nipam patel lab, Excellent resource for antibody staining in Drosophila embryos and larvae from Nipam patel lab