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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
The detection and visualisation of proteins with immunolabelling involves, broadly speaking, three key steps: Tissue preparation (sectioning of muscle tissue sample or fixation of cells in-vitro).Immunolabelling of proteins of interest using antibodies.Detection and visualisation of the protein of interest using fluorescent microscopy.
Heterogeneity Among CD36+ Cells in Normal and Diseased Human Skin
Published in Brian J. Nickoloff, Dermal Immune System, 2019
Immunolabeling was performed with representative antibodies from each of the categories listed in Table 2, except for factor XHIa, Mo3e, RFD1, and those from Group 11D. Single immunolabeling utilized either an indirect immunofluorescence (IIF) or a peroxidase-conjugated ABC (streptavidin-bio-tin complex) technique, as previously reported.57,59 In the latter method, 3-amino-9-ethylcarbazole (AEC) was used as the peroxidase substrate, and the sections were lightly counterstained with Mayer’s hemalum. In every staining procedure, irrelevant MAbs of the appropriate Ig isotype and protein concentration were used as negative controls. Unless otherwise indicated, phosphate-buffered saline and 10% normal goat serum were used for the washing and blocking steps, respectively.
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
Immunolabeling is one of the most useful and used special techniques available to nonclinical pathologists. Because immunolabeling uses antibodies to distinguish particular antigens on the surface or within cells, it is a highly specific method for identifying particular cells, cell components, or molecules of interest beyond what routine H&E or special histochemical staining can (see Figures 6.1c and d and 6.2a–c). Although antibodies usually target structural proteins, antibodies can also be generated against soluble proteins such as enzymes, hormones, and neurotransmitters. This extremely sensitive and specific identification method can provide important information about test article effects and pathogenesis in nonclinical safety assessment. IHC can also be used to help classify poorly differentiated neoplasms and diagnose metastases of uncertain origin. IHC is best used to further characterize or refine an H&E morphologic diagnosis and should include proper controls and use panels instead of single antibodies whenever possible.
Mapping densely packed αIIbβ3 receptors in murine blood platelets with expansion microscopy
Published in Platelets, 2022
Hannah S. Heil, Max Aigner, Sophia Maier, Prateek Gupta, Luise M.C. Evers, Vanessa Göb, Charly Kusch, Mara Meub, Bernhard Nieswandt, David Stegner, Katrin G. Heinze
The higher resolution achieved by 10x expansion comes at the cost of signal dilution, a longer and harsher sample preparation and a decreased mechanical stability of the hydrogel. To address the issue of signal loss and dilution, several modifications of the anchoring chemistry and homogenization procedure have been reported to allow post-expansion labeling [37–39]. For post-expansion immuno-labeling the respective epitopes have to be retained in a recognizable state during sample preparation. The specific platelet antibodies used here did not fulfill this major prerequisite. Therefore, we used direct immunolabeling to minimize the linkage error. Based on our simulations, however, the linkage error is not the dominant limiting factor here, while the unfavorable label retention ratio significantly reduced the maximum degree of colocalization in the image.
Neuroprotective effects of food restriction in a rat model of traumatic brain injury – the role of glucocorticoid signaling
Published in Nutritional Neuroscience, 2022
Milka Perović, Milena Jović, Smilja Todorović, Aleksandra Mladenović Đorđević, Desanka Milanović, Selma Kanazir, Nataša Lončarević-Vasiljković
For IHC analysis, rat brains were prepared as previously describe [15]. The sections were incubated in PBS overnight at 4°C with rabbit polyclonal anti-Ser211 GR antibody (1:50, Cell Signaling #4161S), and after rinsing, with the AlexaFluor-555 labeled goat-anti-rabbit IgG (Invitrogen). Following counterstaining with SYTOX® green fluorescent nuclear and chromosome nucleic acids staining, the sections were mounted in fluorescent mounting medium (Dako). To test the specificity of the immunolabeling, the primary antibody was omitted in control experiments. All the photomicrographs were obtained using Leica TCS SP5 Confocal Laser Scanning Microscope (Leica Microsystems) equipped with Argon 488 nm and NeHe543/633 nm lasers using 20×/1.30 lens. Emission of fluorescently labeled p-GR and SYTOX® green were collected sequentially. All images were taken with a 512 × 512 pixel resolution and 8-bit color depth. For the purpose of colocalization analysis three sections per animal, from three animals were used. The tissue that surrounds lesion (in the zone of 200 µm from the lesion border) was used as the region of interest for further analysis. Colocalization analysis was performed in ImageJ software (NIH, USA) and the degree of colocalization between p-GR and SYTOX® green was measured using the Pearson correlation coefficient (PCC). PCC measures the strength of a linear relationship between fluorescent intensities from the two images and produces values ranging from 1 (perfect positive correlation) to −1 (perfect inverse correlation), with 0 representing a random distribution [18].
Enhanced detection of ATTR amyloid using a nanofibril-based assay
Published in Amyloid, 2021
M. Mahafuzur Rahman, Benjamin Schmuck, Henrik Hansson, Torleif Härd, Gunilla T. Westermark, Mats Sandgren
Steric hindrance prevents the antibody from simultaneous binding to all epitopes on a polyvalent antigen, where binding epitopes are closely spaced on the surface [31]. Thus, the addition of excess antibody in an immunolabeling experiment does not improve the reactivity. For immunolabeling experiments, lower antibody concentration is often preferred. However, lower antibody concentration may generate very weak signals, especially when targeting small antigen deposits (aggregates). Therefore, linking an additional antibody to the antigen, by any means, could offer a valuable tool in immunolabeling experiments. With the use of Ab-bNF, we succeeded in introducing 10-times more primary antibody to amyloid deposits on tissue sections, which resulted in very strong signals, indicating that our concept nicely tackles the steric hindrance issue. Moreover, our enhanced method demonstrated the potential to amplify fluorescence signals from minute amyloid deposits in tissue.