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Immunohistochemistry uses monoclonal antibodies to detect specific proteins in tissue samples with a visible label. For this practical, immunohistochemistry was used to visualise the distribution of protein in the 3rd instar Drosophila melanogaster larvae brain. The distribution of protein was visualised using a primary monoclonal antibody, and this primary antibody was then detected by a secondary antibody. Detection of the secondary antibody is via an enzyme reaction which produces a coloured precipitate. The secondary antibody used is a monoclonal antibody that recognises IgG from a mouse which is attached to alkaline phosphatase. Looking at the protein distribution pattern in the larval brain the antibody used was detected. Brain samples were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) at room temperature for 20 minutes, then washed four times with PBST prior to blocking with 10% donkey serum for 45 minutes. The primary antibody was added, and the brains incubated at 4°C overnight. The antibody used was embryonic lethal abnormal vision (ELAV). This is because there are domains in the optic lobe which look like dots and webs from binding of ELAV to the specific proteins in the brain.
Immunohistochemistry is the use of monoclonal and polyclonal antibodies to detect specific proteins in sections of tissues. Applications of immunohistochemistry has been used by pathologists for the diagnosis of cancers. This is because certain antigens particularly tumour antigens are expressed in certain cancers (Kaliyappan et al, 2012). The main principles of immunohistochemistry include a primary antibody which binds to a specific protein of interest. Then a complex of an antibody binding to a specific protein is formed from incubation with a secondary antibody. Finally, in the presence of a substrate and chromogen, an enzyme catalysis happens to generate a coloured precipitate at the antibody-protein specific binding sites (Immunohistochemistry.us, n.d.). Monoclonal antibodies are specific antibodies produced by a B lymphocyte cell which recognises a specific protein such as an antigen. Polyclonal antibodies on the other hand are antibodies produced by a collection of different B lymphocytes. They are able to recognise specific epitopes on an antigen (Lipman et al., 2005). Primary antibodies are antibodies which bind to a specific antigen. Monoclonal and polyclonal antibodies are classified as primary antibodies (Cheriyedath, 2016). Secondary antibodies are the antibodies which bind to the primary antibody to allow detection of the primary antibody under a microscope (Rockland-inc.com, n.d.). Primary and secondary antibodies are used because primary antibodies detect a complimentary protein which is of particular interest whilst the secondary antibody is raised against immunoglobulins associated with the primary antibody. The secondary antibody then associates with a linker molecule which then recruits reporter molecules or the antibody binds directly to the reporter molecule. Drosophila is used as a model organism because their genome is very closely related to the human genome. This means that human genes can be mapped to drosophila genes. Genes in humans which cause disease are also in drosophila. Many generations can be observed in drosophila because they have a short reproductive cycle. They are small so a lot of them can be kept in a laboratory. Fruit fly genes can be altered very easily. Transgenics can be easily produced in the flies and studied. A huge amount of biology is known for these fruit flies due to a lot of research and history in these flies (Jennings, 2011). Tumour-associated stromal cells can act non-autonomously to produce epithelial cancers. Using the drosophila model organism mutations have been identified in the human tumor susceptibility gene 101 (Tsg101) as a cause of these cancers. The Tsg101 homolog leads to Notch signalling and secretion of the JAK-STAT ligand, making neighbouring cells to grow. Drosophila serves as a model for cancer. The most present malignancies in the central nervous system include gliomas. Pathways for the gliomas in drosophila using the GAL4 system causes glias to increase and damage brain cells. (Spradling et al, 2006). The aim of this experiment was to use immunohistochemistry in order to detect protein distribution in the brain of the third instar Drosophila melanogaster larvae. By analysing the immuno stained sample the primary antibody used can be detected.
Steps of immunohistochemistry include: tissue preparation by fixation, protein retrieval, endogenous blockage and antibody labelling. In tissue preparation, tissues samples are preserved so protein and tissue architecture does not breakdown. Samples may be also need to be perfused for prevention of detecting non-specific proteins which can interfere with the detection of the target protein. In order for protein and tissue to not breakdown fixation is required. Steps of fixation include perfusion, immersion and freezing. In perfusion, tissues are perfused with a fixative to allow rapid fixation. During immersion tissues are immersed in the fixative to allow the fixative solution to diffuse through the tissue or cell sample. In the final step samples with protein that cannot survive fixation are frozen in liquid nitrogen whilst being embedded in a cryoprotective embedding medium. Once fixation is done the sample needs to be sectioned and mounted. The tissues which have been fixed with a paraformaldehyde solution are sectioned into slices which measure 4-5µm using a microtome. After sectioning the sections are mounted onto a glass slide which is coated with an adhesive. Once mounting has been done the sections are dried and then deparaffinization takes place (Thermofisher.com, n.d.). The next major step is for the target protein to be retrieved. Paraffin is removed in order for the antibodies to reach the protein. Heat induced epitope retrieval is the most common used method to retrieve these proteins. This involves heating the slide at PH6 or PH9 depending on the antibody (Biosciences, 2014). Endogenous blocking needs to place because reagents which are present during immunohistochemistry are also present in cells. This can interfere with protein detection because of unwanted signals being produced that mistakenly detect an protein. Finally, the antibodies are labelled to detect the target antigens. The primary and secondary antibodies are diluted into a buffer in order for the antibody to be stabilised. Rinse buffers are used between antibody applications to remove unbound antibodies (Thermofisher.com, n.d.). Once they are labelled the samples are viewed under a microscope (Biosciences, 2014).
Summary flow chart
The primary antibody based on the protein distribution in the drosophila brain sample is the embroyonic lethal, abnormal vision (ELAV).
A`ntenna - A pair of appendages used for sensing.
Eye disc - The raised disk on the retina at the point of entry of the optic nerve, lacking visual receptors and so creating a blind spot.
Optic stalk - The constricted proximal portion of the optic vesicle in the embryo which forms the optic nerve.
Optic lobe - The lobe in the midbrain from which the optic nerve partly arises.
Ventral nerve cord - A chain of connected segmental ganglia which lies against the body wall in the body of the drosophila.
In this practical, distribution of protein in the 3rd instar Drosophila melanogaster larvae brainwas detected using a primary antibody which binds to the specific antibody. Using a secondary antibody, the protein distribution was able to be visualised under a microscope by binding to the primary antibody. Based on the protein distribution visualised under the microscope the primary antibody used to bind to the proteins to produce the protein distribution was identified. The identified antibody was ELAV because there are domains in the optic lobe which look like discrete dots and webs.
Embryonic lethal abnormal vision protein (ELAV protein) is a protein which binds to RNA thus an RNA binding protein expressed in brain cells of drosophila after birth. Repeats of an RNA binding domain approximately 80 amino acids in length is in these RNA-binding proteins. ELAV distribution has a similar distribution to many different splicing factors. Variants of genes which are correctly spliced in neurones are regulated by this ELAV protein. Additionally, this ELAV protein plays a role in the formation of the n-arm transcript of the drosophila armadillo. It is generated by splicing from exclusion from exon six of the ubiquitous arm. In mutant ELAV the amount of n-arm is reduced (Broody, 1996). Mutant ELAV have a short life span (Toba et al, 2010). Loss of function alleles for ELAV causes the drosophila embryo to be lethal with also mutant embryos having an abnormal neuropil. Also mutations produce abnormal eye structures, defective electroretinograms and flight defects. Mutant clones analysed in mosaic flies has shown that there is a post-embryonic role for ELAV for photoreceptor cells, optic lobe and associated neuropil areas to maintained. Because of this, phenotypes that appear to be a mutant has a role for ELAV in the formation and maintenance of the nervous system (Koushika et al, 1996).
Significant divergence between humans and mouse has caused orthologous disease genes to arise. Orthologous genes arise due to speciation in homologous genes. An example is the TDP1 gene. This is a gene which has a role for Topo I DNA complexes to be repaired. TDP1 orthologs for expression in the inside of cells with localizations has been located in the cytoplasm and nucleus. Mutations in the TDP1 gene has been seen to have a causative link to spinocerebellar ataxia with axonal neuropathy (SCAN1) disorder. This mutation is not present in mouse ortholog. TDP1 expression in human and mouse are different and produce different phenotypes. Selectins P and E for inflammation is different when compared between humans and mouse. The orthologous human version of selectin P from mouse does not have the pathway needed for regulation (Gharib and Robinson-Rechavi, 2011).
Immunohistochemistry is a useful technique because it is used in research and pathology laboratories where immunohistochemistry can help with diagnosing neoplasias and pseudo-neoplastic lesions (De Matos et al, 2010). IHC is used to test efficacy of pharmaceutical drugs by detecting the activity or the regulation of disease targets. Immunohistochemistry is a technique specifically for detecting proteins such as antigens in a tissue sample. In situ hybridisation involves finding the location of targets for specific nucleic acids in tissues and cells which have been fixed to gain information for expression of genes and genetic loci (Ncbi.nlm.nih.gov, n.d.). RT-PCR is used for RNA expression analysis. Knowing the mRNA is important to understand what gene it is and protein distribution allows us to understand what affect the gene has in an organism.
1) The point of fixation is to fix the tissue sample so that putrefaction and autolysis does not occur. Fixation allows the tissue sample to kept in its natural state. This is important so that the structures in the cell do not fall apart and diffuse away. The fixative used in fixation disables intrinsic biomolecules, protects the tissue from extrinsic damage and also alters the tissue on a molecular level to increase their mechanical strength and stability. The step is very critical because proteins need to be localized because otherwise they will diffuse away from their initial location. Protein immobilization occurs before translocation. The final thing for fixation is that it achieves the most rapid reaction possible at a low temperature (Berod et al, 1981).
2) Antibodies bind to epitopes on their complementary antigen. However sometimes these antibodies can bind to non-specific antibodies that is similar to the binding site on the target antibody. Because of this binding of non-specific sites, background staining can be created that can mask the detection of the target protein. To get rid of this background staining tissue samples are incubated with a buffer that blocks the reactive sites which are not 100% complimentary to the primary and secondary antibodies (Thermofisher.com, n.d.).
3) NTMT solution is a buffer used for the blockage of non-specific binding sites (Ebioscience.com, n.d.). The colour reaction is the final step which involves visualising the tissue sample where a specific antibody binds to the tissue sample. After this the primary antibody is detected in two ways, directly or indirectly. The direct method is the colour producing reaction. The primary antibody is tagged with a peroxidase enzyme which is used in a reaction to generate a coloured product (Heyderman, 1979).
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