Optimization in immunohistochemistry is often a time consuming and complex process. There are a varying array of moving parts to consider all while preserving the sensitivity and specificity of the test. When optimizing an antibody it is important to consider the fixation of the tissue and the type of epitope retrieval that would be best suited for the test. The dilution of the primary antibody is a key marker for the efficiency and effectiveness of the laboratory protocol. The purpose of this study was to produce an optimized antibody for the nuclear protein in testis to detect NUT midline carcinoma that provides a sensitive and specific test but is also efficient and can be useful for everyday pathological dedications. The midline carcinoma defined by the translocation of the NUT gene on chromosome 15q14 that bonds with BRD4 or BRD3 commonly known as NUT midline carcinoma (NMC) is a rapidly aggressive and fatal disease. Commonly a fluorescent in situ hybridization (FISH) test is used to diagnosis this carcinoma. This test takes longer than traditional IHC and can delay the treatment of the patient. Therefore this is why, irrespective of the levels of tumor markers, immunohistochemistry for the NUT marker should be performed in any case where there is poorly differentiated carcinomas that do not have glandular differentiation that come from midline structures.