Explore chapters and articles related to this topic
Immunochemistry of Cell Wall – a Tool for Evaluation of the Response of Plants to Changed Habitat
Published in Artur Dyczko, Andrzej M. Jagodziński, Gabriela Woźniak, Green Scenarios: Mining Industry Responses to Environmental Challenges of the Anthropocene Epoch, 2022
Katarzyna Sala, Kamila Godel-Jędrychowska, Ewa Kurczyńska
Immunochemistry (ICH) includes immunocytochemistry (ICC) which examines cells and immunohistochemistry (IHC) which examines the tissues. It is a method that allows for identification or localisation of specific antigens within cells or tissues. The method utilises an antigen-specific antibody which binds an antigen in a cell/tissue section, fluorochromes (at the light microscope level) and colloidal-gold particles (at the electron microscopy level) to visualize where the antibody was bound. ICH, nowadays, is a common laboratory technique that is used to visualize the presence of a specific antigen in cells/tissues by use of a specific primary antibody that binds to it. In a two-step reaction, the primary antibody allows for visualization of the antigen under a fluorescence microscope only when it is bound by a secondary antibody that has a conjugated fluorophore (Fig. 2). ICH helps to evaluate whether or not cells/tissues in a particular sample express the antigen in question. In cases where an immunopositive signal is found, we are able to determine which sub-cellular compartments are expressing the antigen.
Targeted Systemic Combinatorial Delivery of siRNA Polyplexes–Functional Quantum Dot-siRNA Nanoplexes
Published in Loutfy H. Madkour, Nanoparticle-Based Drug Delivery in Cancer Treatment, 2022
Water-soluble QDs, which are often applied to label biological molecules, may be an excellent choice. QDs exhibit strong fluorescence and can pass through the cell membranes and blood-brain barrier [101]. The toxic effect of CdSe QDs can be improved by modification such as the incorporation of a ZnS shell and polyethylene glycol (PEG) coating [102,103]. Previously, QD-based siRNA delivery was usually achieved by mixing QDs with transfection agents such as PEI or combined with another class of nanomaterial [97,104]. However, the addition of other agents may lead to vulnerability to intracellular degradation or increased cytotoxicity. An alternative is the use of nonselective or selective bioconjugation techniques. Selective bioconjugation can occur without a preceding reduction reaction, thus retaining the integrity of the siRNA [105]. The use of a heterobifunctional crosslinker such as sulfo succinimidyl-4-(N-maleimido methyl)cyclohexane-1-carboxylate (sulfo-SMCC) results in selective bioconjugation toward specific sites on the protein to form a stable thioether bond with a sulfhydryl-exposed antibody [106]. QD-SMCC has been applied in immunohistochemistry only for antibody bioconjugation. The potential for gene delivery, particularly in the study of MSC differentiation, has yet to be sufficiently investigated.
Immunofluorescence
Published in Guy Cox, Fundamentals of Fluorescence Imaging, 2019
Of the chemical fixatives, 4% formaldehyde in phosphate buffer is the most common. Formaldehyde crosslink proteins and thus stabilizes cell membranes and organelles, forming a lattice that permits diffusion of various substance including antibodies into cells. Aldehyde fixation is a prerequisite for immunohistochemical detection of soluble proteins, including neurotransmitters, neuropeptides, calcium-binding proteins and reporter proteins such as eGFP, to avoid washout of damaged cells during the staining procedure. However, loss of antigenicity and/or epitope masking that are due to tissue fixation are major limitations in immunohistochemistry [26]. It is appropriate to apply this to both enzyme and fluorescent immunohistochemistry since both techniques are affected equally. Clevenger et al. [27] have shown that the sequential use of formaldehyde and 0.1% Triton X-100 results in optimal permeabilization of cells for the demonstration of nuclear antigens.
Efficacy of polyvinylpyrrolidone-capped gold nanorods against 7,12 dimethylbenz(a)anthracene-induced oviduct and endometrial cancers in albino rats
Published in Egyptian Journal of Basic and Applied Sciences, 2023
Hend Gamal, Walid Tawfik, Hassan H El-Sayyad, Heba Mohamed Fahmy, Ahmed N. Emam, Heba A El-Ghaweet
In the oviduct, Ki-67 immunohistochemistry revealed higher immunostaining in the DMBA carcinogenesis group, indicating more unusual proliferative activity (Figure 10d,e). However, when the DMBA carcinogenesis group was treated with PVP-capped AuNRs, the Ki-67 immunohistochemical reactivity was reduced (Figure 10f). Following image analysis, Ki-67 showed that the immune response to DMBA carcinogenesis was significantly higher than in the other groups that had been studied (Figure 8). Also, the uterine endometrium of negative control possessed weak-to-moderate positive reaction for Ki-67 (Figure 11a) compared to more reaction post-DMBA carcinogenesis injection (Figure 11c,d). In contrast, improved weak reaction was remarked in DMBA carcinogenesis treated with PVP-capped AuNRs (Figure 11e,f). Ki-67 displayed an essential increase in the immune reaction in the DMBA carcinogenesis, improved in DMBA carcinogenesis treated with PVP-capped AuNRs group but still did not match with control values (Figure 12).
Effect of polycaprolactone scaffolds containing different weights of graphene on healing in large osteochondral defect model
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Ozgur Basal, Ozlem Ozmen, Aylin Muyesser Deliormanli
Selected sections were immunostained with BMP-2 (anti BMP-2 antibody (ab6285) Abcam, Cambridge, UK), kollagen 1 (Collagen 1 antibody; ab34710, Abcam, Cambridge, UK), ALP (Anti-ALP antibody; ab67228, Abcam, Cambridge, UK) and VEGF (Anti-VEGF antibody bs-1957R), 1/100 dilution by streptavidin biotin technique. The sections were incubated with the primary antibodies for a period of 60 min. The immunohistochemistry was performed using a biotinylated secondary antibody and a streptavidin–alkaline phosphatase conjugate. The EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC kit (ab80436) was used as the secondary antibody. The antigens were demonstrated using diaminobenzidine (DAB) as the chromogen. For negative controls, the primary antiserum step was omitted. All examinations were performed on blinded samples. All the slides were analyzed for immunopositivity and to evaluate percentage of immune-positive cells for each marker. Seven serial sections were prepared and analyzed for each rabbit, and 2 areas of each section were scored semi-quantitatively by considering staining intensity (0, absence of staining; 1, slight; 2, medium and 3, marked). Statistical analyses were subjected to the results obtained from the image analyzer. Morphometric analyses were performed using the Database Manual Cell Sens Life Science Imaging Software System.
Mitochondrial uncoupling protein 2 is regulated through heterogeneous nuclear ribonucleoprotein K in lead exposure models
Published in Journal of Environmental Science and Health, Part C, 2020
Gaochun Zhu, Qian Zhu, Wei Zhang, Chen Hui, Yuwen Li, Meiyuan Yang, Shimin Pang, Yaobing Li, Guoyong Xue, Hongping Chen
Antibodies and reagents were obtained commercially as indicated below, including lead acetate (PbAc) (Tianjin benchmark Chemical Reagent Co., Ltd), rabbit anti-UCP2 polyclonal antibody (Bioss company), rabbit anti-hnRNP K polyclonal antibody (Abcam company), mouse anti-β-actin polyclonal antibody (Sigma company), goat anti-rabbit and goat anti-mouse secondary HRP-conjugated antibody (Sigma company), biotin goat anti-rabbit IgG (Boster company), streptavidin-peroxidase (Boster company), TRITC labeled goat anti-rabbit IgG (ImmunoResearch Laboratories Jacksonand), and lipofectamine 2000 (Genepharma company). Rabbit anti-UCP2 polyclonal antibody was used at 1:500 for western blotting, and 1:100 for immunohistochemistry and immunofluorescence. Rabbit anti-hnRNP K polyclonal antibody was used at 1:1000 for western blotting, and 1:500 for immunohistochemistry and immunofluorescence. And mouse anti-β-actin polyclonal antibody was used at 1:1000 for western blotting. Vector and GFP-hnRNP K were kind gifts from Professor Gao XueJuan (from Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, P. R. China.).