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Histological Study in Orthopaedic Animal Research
Published in Yuehuei H. An, Richard J. Friedman, Animal Models in Orthopaedic Research, 2020
Helen E. Gruber, Audrey A. Stasky
Other common problems encountered with immunohistochemistry are poor visualization resulting from insufficient specific staining, high levels of nonspecific (background) staining, the inability to achieve localization due to masking by prolonged fixation or embedding methods, and lack of success with initial trials of new antibodies. Thorough rinsing between steps is important to control non-specific staining. Some investigators have reported success in retrieving antigenicity in specimens fixed for inappropriately long periods (or older archived fixed tissue) with use of antigen retrieval methodologies as recently reviewed elsewhere.33-37 These methods include techniques such as enzyme pretreatment of sections and microwave irradiation.
Routine and Special Techniques in Toxicologic Pathology
Published in Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard, Toxicologic Pathology, 2018
Daniel J. Patrick, Matthew L. Renninger, Peter C. Mann
Epitope masking by formalin protein cross-linking can mostly be removed by the use of heat-induced epitope retrieval (HIER). HIER was one of the most revolutionary technical advances in the evolution of IHC as it greatly increased the ability to detect antigens in formalin-fixed tissues. HIER has decreased the need for frozen sections or coagulating (non-cross-linking) fixatives such as ethanol. Microwave oven, pressure cooker, and steam are the most common heating methods; autoclave and water bath are also sometimes used. HIER can also be modified by varying the types of buffers used and varying the pH of the buffers. Antigen retrieval can also be achieved by enzymes (e.g., proteinase K, trypsin, pepsin), alone or more commonly as a pretreatment before HIER. Certain methods can be harsher than others, and overall, there is no best retrieval method for every antigen. The degree of background staining increases proportionally to the intensity of antigen retrieval. Since fixation times are often short in nonclinical studies, HIER is sometimes unnecessary. HIER can also be used to unmask antigens in semithin sections of tissue that have been embedded in methyl methacrylate (MMA) after thorough deplasticization, which allows for various immunohistochemical evaluations of MMA plastic-embedded tissue (Hand and Church 1998).
Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
Following fixation and embedding (especially with paraffin), insufficient signal may be seen with immunostaining, as epitopes may be obscured. Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. To overcome this limitation, several methods of “antigen retrieval” have been developed. The choice of which specific method to use is dependent on both the antibody used andthe epitope being analyzed.
Corosolic acid inhibits tumour growth without compromising associating liver partition and portal vein ligation-induced liver regeneration in rats
Published in Annals of Medicine, 2022
Jinwei Zhao, Weiyi Zhao, Hongyue Xu, Wenjing Luan, Xuefei Wang, Yimu Fang, Lu Yu
The fixed tissue samples were embedded in paraffin. The blocks were sectioned at a thickness of 4 µm and then de-paraffinized. The dewaxing and rehydration of the sections were performed using the following procedure. The sections were incubated in xylene for 20 min, followed by decreasing the concentrations of ethanol (100%, 95%, and 75%), and were then washed with double-distilled water. Antigen retrieval was performed using the manufacturer’s buffer. The sections were blocked using 3% hydrogen peroxide and phosphate-buffered saline containing 5% bovine serum. Primary antibodies against cluster of differentiation (CD) 8 (ABclonal, A11856), CD86 (ABclonal, A10795), CD206 (ABclonal, A8301), CD31 (ABclonal, A0378), Ki67 (ABclonal, A11390), TGF-β (ABclonal, A10749), and PD-1 (ABclonal, A11973) were used in this study.
Current techniques to accurately measure anti-retinal autoantibodies
Published in Expert Review of Ophthalmology, 2020
Antibody binding to retinal cells can be also examined by more sensitive methods such as immunofluorescent IHC on frozen retinal slides [23]. Detection of binding is localized by commercially available secondary antibodies labeled with a fluorescent dye and examined in a fluorescent or confocal microscope [22,25]. In both the colorimetric and fluorescent methods, labeling to retinal layers, such as rods and cones in photoreceptor cell layer, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, and ganglion cell layer can be evaluated by intensity of the color or of the fluorescence, respectively. Patterns of immunostaining of human retina by different serum AAbs is usually dependent on antibody fine specificity [23]. The major disadvantage of detection of AAbs by IHC technique is that this methodology alone is not sufficient for AAb identification, and has to be followed up by the confirmation of specificity by western blotting or other, more sensitive methods. Another limitation of this method is that some AAbs cannot access an antigen in the tissue, producing a negative result and subsequently, special manipulations are required, e.g. antigen retrieval. Preservation of tissue with formaldehyde can change protein chemistry and antibodies do not bind to antigenic epitopes. Antigen retrieval refers to any technique that reverse masked epitope and antibody binding is restored. Two methods are frequently used, protease-induced epitope retrieval and heat-induced epitope retrieval.
Juxtaposition of IL-1β and IFN-γ expression and apoptosis of keratinocytes in adult-onset Still’s disease
Published in Expert Review of Clinical Immunology, 2019
Shijia Rao, Qianwen Li, Haijing Wu, Ming Zhao, Alun Wang, Guiying Zhang, Ji Li, Lixia Lu, Wei Shi, Qianjin Lu
Skin biopsies were fixed in formalin, embedded in paraffin and sequentially sectioned (3 μm thick). Regular hematoxylin and eosin slides were prepared. Antigen retrieval was carried out using retrieval solution (Servicebio, CN). Before using 5%BSA (Well-Biology, CN) blocking solution to block nonspecific binding, periodic acid and peroxidase blocking solution were also used to decrease nonspecific binding. Anti-CD3, anti-CD4, anti-CD8 (Maxim, CN), anti-CD11b, anti-CD19, CD103, anti-IFN-γ, anti-IL17, anti-ferritin heavy chain (Abcam), CD69, anti-IL4, anti-IL6, anti-IL1β (Proteintech, NJ, USA), anti-CD68 (CST), and anti-IL18 (Sigma) antibodies were used as primary antibodies in our work. The secondary antibody and staining were from an Opal 7-color IHC detection kit (PerkinElmer, Hopkinton, MA). 4ʹ,6-diamidino-2-phenylindole (DAPI) make cell nuclei visible. Skin slides were scanned by the PerkinElmer Vectra, and images were analyzed using inForm software (PerkinElmer, Hopkinton, MA).