Detection Systems in Immunohistochemistry

Immunohistochemistry (IHC) is a process of selectively imaging antigens in cells or tissue sections by exploiting antibody specificity. This technique is widely used in diagnostic pathology and research experiments for tracking specific molecular markers characteristic of a particular cell type or cellular events such as cancerous cell development, cell proliferation, or apoptosis. Visualizing the target antigen following an antibody-antigen interaction is accomplished by different detection systems. In the simplest instance, primary antibody directly conjugated to an enzyme is responsible for both specifically binding to the antigen and catalyzing a color-producing reaction. Alternatively, complex detection systems could be designed to profoundly improve minimal detection level of the antigen. During the past years, there has been a considerable improvement in designing and introduction of new and highly sensitive detection systems. The choice of an IHC detection system is a compromise of a variety of variables including desired sensitivity, cost, and the time needed for an IHC staining to be performed. This chapter covers the immunohistochemistry detection systems with emphasis on their principle, history, advantages, and limitations and delineates factors needed to be considered for choosing an appropriate detection system for IHC applications

Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

<p>Abstract</p> <p>Background</p> <p>Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His) tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in <it>Escherichia coli </it>and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices.</p> <p>Results</p> <p>Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel) for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel) did not work well with respect to specific binding capacity and recovery profile.</p> <p>Conclusions</p> <p>Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also provide helpful hints for those researchers facing same challenge.</p

Culture density of menstrual blood-derived stromal/stem cells determines the quality of T cell responses: An experimental study

Background: Menstrual blood-derived stromal/stem cells (MenSCs) are a new population of refreshing and highly proliferative stem cells. Immunomodulatory effects of MenSCs profoundly depend on their relative density. Objective: To find whether MenSCs cultured at varying numbers would differentially affect the allogenic peripheral blood mononuclear cells (PBMCs) key features. Materials and Methods: PBMCs were co-cultured with various MenSCs numbers. PBMCs proliferation was investigated via 3H-thymidine incorporation. Flow cytometry was used to assess human leukocyte antigen (HLA)-DR, HLA-ABC, HLA-G, and costimulatory markers on MenSCs and the percentage of regulatory T cells (Tregs) among PBMCs. The concentration of cytokines was determined in supernatant of co-cultures. Results: The support of PBMCs proliferation at low MenSCs densities correlated with higher levels of pro-inflammatory interferon gamma (IFN-γ) in MenSCs/PBMCs co-culture and increased expression of HLA-DR by MenSCs. On the other hand, the suppressive property of MenSCs at higher densities was independent of Treg frequency, but correlated with a high concentration of Interleukin (IL)-6 and IL-10 in the co-cultures. Conclusion: Totally, at different seeding densities, MenSCs could differentially interact with PBMCs leading to significant changes in the level of anti- and/or pro-inflammatory factors. These preliminary in vitro results are suggested to be taken into consideration in experimental models of MenSC-based immunomodulation. Nonetheless, for efficient utilization of MenSCs anti-inflammatory features in pre-clinical disease models, we still need to broaden our knowledge on MenSC-immune system cross-talk; this could play a part in designing more optimized MenSCs injection modalities in the case of future pre-clinical and subsequently clinical settings. Key words: Menstrual, Stromal cells, T cell response, Interferon

Overexpression and translocation of dynamin 2 promotes tumor aggressiveness in breast carcinomas

Dynamin 2 is a GTPase protein that has been implicated in cancer progression through its various roles such as endocytosis, morphogenesis, epithelial-mesenchymal transition (EMT), cellular contractions, and focal adhesion maturation. The increased expression levels of this molecule have been demonstrated with the development of several cancers such as prostate, pancreas, and bladder. However, its clinical significance in breast cancer is unclear yet. In the present study, the membranous, cytoplasmic, and nuclear expression levels of dynamin 2 molecule were evaluated for the first time, using immunohistochemistry (IHC) on tissue microarray (TMA) slides in 113 invasive breast cancer tissues. Moreover, afterward, the association between the dynamin 2 expression and clinicopathological features was determined. Our finding showed that, a higher nuclear expression of dynamin 2 is significantly associated with an increase in tumor stage (P = 0.05), histological grade (P = 0.001), and age of the patients (P = 0.03). In addition, analysis of the cytoplasmic expression levels of this molecule revealed that, there was a statistically significant difference between the expression levels of dynamin 2 among the different breast cancer subtypes (P = 0.003). Moreover, a significant association was found between the increased expression of dynamin 2 membranous and vascular invasion (VI) (P = 0.02). We showed that dynamin 2 protein expression has an association with more aggressive tumor behavior and more advanced disease in the patients with breast cancer; therefore, dynamin 2 molecule could be considered as an indicator of disease progression and aggressiveness

Conjugation of R-Phycoerythrin to a Polyclonal Antibody and F (ab&apos;)2 Fragment of a Polyclonal Antibody by Two Different Methods

Abstract R-Phycoerythrin (R-PE), a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F(ab&apos;)2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry (ICC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity

Placenta:an old organ with new functions

The transition from oviparity to viviparity and the establishment of feto-maternal communications introduced the placenta as the major anatomical site to provide nutrients, gases, and hormones to the developing fetus. The placenta has endocrine functions, orchestrates maternal adaptations to pregnancy at different periods of pregnancy, and acts as a selective barrier to minimize exposure of developing fetus to xenobiotics, pathogens, and parasites. Despite the fact that this ancient organ is central for establishment of a normal pregnancy in eutherians, the placenta remains one of the least studied organs. The first step of pregnancy, embryo implantation, is finely regulated by the trophoectoderm, the precursor of all trophoblast cells. There is a bidirectional communication between placenta and endometrium leading to decidualization, a critical step for maintenance of pregnancy. There are three-direction interactions between the placenta, maternal immune cells, and the endometrium for adaptation of endometrial immune system to the allogeneic fetus. While 65% of all systemically expressed human proteins have been found in the placenta tissues, it expresses numerous placenta-specific proteins, whose expression are dramatically changed in gestational diseases and could serve as biomarkers for early detection of gestational diseases. Surprisingly, placentation and carcinogenesis exhibit numerous shared features in metabolism and cell behavior, proteins and molecular signatures, signaling pathways, and tissue microenvironment, which proposes the concept of "cancer as ectopic trophoblastic cells". By extensive researches in this novel field, a handful of cancer biomarkers has been discovered. This review paper, which has been inspired in part by our extensive experiences during the past couple of years, highlights new aspects of placental functions with emphasis on its immunomodulatory role in establishment of a successful pregnancy and on a potential link between placentation and carcinogenesis.</p

An Efficient One-Pot Multicomponent Synthesis of 4-Aza-Podophyllotoxin Derivatives in Ionic Liquid

A simple, green, and efficient procedure for the synthesis of 4-aza-podophyllotoxin derivatives by using a one-pot three-component reaction of benzaldehydes, 1,3-cyclohexanediones, and anilinolactones in the presence of catalytic amount of alum in 1-butyl-3-methylimidazolium triflate as green media is described. This reaction proceeded under mild conditions with the use of an inexpensive and readily available catalyst, high to excellent yields, and simple workup procedure

Expression, Purfication and endotoxin removal of Brucella melitensis DnaK and Omp31 proteins

Background: New strategies are needed to protect against Brucella melitensis infection. Subunit vaccines offer a promising approach because they can stimulate both cellular and humoral immunity, so high production of recombinant protein with less content of endotoxin is desired. In present study, we described a method for expression and purification of B.melitensis recombinant DnaK(rDnaK) and Omp31(rOmp31) proteins while less content of endotoxins were detected in final product. Material and Methods: Recombinant pET-dnak and pDEST-omp31 plasmids were transformed into competent expression host E.coli BL21 (DE3). After induction by IPTG, bacteria were grown at 20◦C for 22h. Then recombinant proteins were purified by Ni-NTA Agarose. Purification was done while two methods using Triton X-114 in washing steps and standard protocol (without detergent) were used in parallel. Results: rDnak and rOmp31 were purified by using Urea. We could obtain 20 and 8 mg recombinant proteins from rDnak and rOmp31 from 1 liter medium, respectively. The amount of endotoxins in final products was less content of 0.05 EU/mg. Furthermore, recovery of protein was up to 80% as compared to the standard protocol. Conclusion: The method used in this study, gives a product with very low extent of endotoxin, but 20% of recombinant proteins were lost. So we think the method described here can be used for purification and endotoxin removal of other recombinant proteins with similar physiologic properties