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Airway Wall Remodelling in the Pathogenesis of Asthma: Cytokine Expression in the Airways
Published in Alastair G. Stewart, AIRWAY WALL REMODELLING in ASTHMA, 2020
Peter Bradding, Anthony E. Redington, Stephen T. Holgate
When used in isolation, immunohistochemistry has a number of limitations. In particular, it gives no information about whether or not the protein identified is being actively synthesised and released, so that simple quantification of numbers of cells expressing immunoreactivity for a particular protein may be misleading. This is illustrated by the observation that numbers of tryptase-immunostaining mast cells are similar in bronchial biopsies from normal subjects and from patients with atopic asthma,8,9 and yet there is clear evidence of mast cell activation in this disease when assessed by measurement of mediators in lavage fluid1,193 or by the ultrastructural appearance of the cells as assessed by electron microscopy.4,8 Thus, although we have been unable to demonstrate a difference between asthmatic and control subjects in the numbers of IL-5- and IL-6-immunoreactive cells in bronchial biopsies,192 this does not exclude the possibilities of increased release and/or increased availability of these cytokines.
Immunohistochemistry of the Pulmonary Extracellular Matrix
Published in Joan Gil, Models of Lung Disease, 2020
Antonio Martinez-Hernandez, Peter S. Amenta
There are many methods and variations available for immunohistochemistry (Martinez-Hernandez, 1987a, b; Amenta and Martinez-Hernandez, 1987; Polak and Varndell, 1984; Bullock and Petrusz, 1982). In any study, choices of fixative sectioning, antibody types, and markers must be based on the possible effects of each treatment on subsequent ones. Therefore, before we present specific protocols, some theoretical considerations are appropriate.
Plaques, Tangles and Amyloid:
Published in Robert E. Becker, Ezio Giacobini, Alzheimer Disease, 2020
Robert G. Struble, H. Brent Clark
Negative results with immunohistochemistry should always be interpreted conservatively since failure to identify an epitope does not mean it is not present. As Wischik et al, (1988) showed, pre-treatment of PHF with pronase modified immunoreactivity by unmasking τ-like immunoreactive sites. Positive staining similarly should be interpreted carefully; an epitope common to several proteins could complicate interpretations of immunoreactivity. For example, an antibody raised against neurofilaments also has been shown to recognize τ, presumably because both proteins have comparable amino acid domains or perhaps slight cross-contamination of the neurofilaments with τ (see Ksiezak-Reding et al, 1987). In sum, there is ample evidence of involvement of cytoskeletal elements in both NFT and PHF. However, the interpretation of studies providing that evidence must be tempered by the realization of the methodologic limitations inherent in immunohistochemistry.
Treatment of copper nanoparticles (CuNPs) for two spermatogenic cycles impairs testicular activity via down-regulating steroid receptors and inhibition of germ cell proliferation in a mice model
Published in Nanotoxicology, 2022
Vanrohlu Nicy, Milirani Das, Guruswami Gurusubramanian, Pradip Mondal, Vikas Kumar Roy
The testicular sections on the slide were cleared and rehydrated. Immunohistochemistry was done according to the previous protocol (Jeremy, Gurusubramanian, and Roy 2019). Tissue slide after processing a dehydration process was kept in PBS for 10 minutes and was incubated with blocking goat serum (1:100 diluted in PBS, Lot# H2114; Santa Cruz, CA, USA) and incubated with primary antibodies overnight at 4 °C. The details of antibodies used for immunohistochemistry has been in Table 1. After incubation, the slide was washed in PBS and probed with HRP-conjugated secondary antibodies (Table 1, goat anti-rabbit IgG secondary antibody and goat anti-mouse IgG anti-mouse secondary antibody for respective primary antibodies) for 4 hours. After incubation, the unspecific binding was washed out with PBS and then incubated in 3’3′-diaminobenzidine tetrahydrochloride hydrate (0.6%) solution in 0.05 M Tris buffer containing hydrogen peroxide. After which it was counter-stained with hematoxylin (for 3β HSD, CPTC-OGG1 and LHR), dehydrated with alcohol, cleared with xylene, mounted with DPX and observed under the microscope. Hematoxylin staining was done for PCNA and GCNA slides, because, PCNA and GCNA are nuclear proteins.
Glucose-regulated protein 78 (GRP78) as a potential novel biomarker and therapeutic target in multiple myeloma
Published in Expert Review of Hematology, 2020
Slavisa Ninkovic, Simon J. Harrison, Hang Quach
The discrepancy in the above findings may be partially explained by the method of assessment of GRP78 expression. Quantification of chromogenic immunohistochemistry relies on the visual estimation of positive staining with significant inter and intra-assessor variability. Multiplex immunohistochemistry, however, is likely to be more accurate as it is an algorithm-driven, machine-learning based, semi-quantitative assessment of expression. In addition, quantification of image H-scores, which was utilized in the latter study, attributes relative weight to more intensely stained cells which is not possible with visual estimation. Furthermore, prognostic correlations in the above studies occurred in different study populations with the one study looking at a cohort of newly diagnosed MM patients while the multiplex IHC was assessed in multiply relapsed patients. It is possible that in the newly diagnosed setting, high levels of GRP78 expression sensitize myeloma cells to proteasome inhibition while in the RRMM setting, despite high abundance of GRP78 expression, cells with highest intensity of GRP78 expression may represent cells where GRP78 interacts with multiple pro-survival ligands leading to cell proliferation and relative resistance to proteasome inhibition. Thus, the role of GRP78 as both a prognostic biomarker and its position as a potential therapeutic target in MM requires further investigation.
Anti-tumor immunity influences cancer cell reliance upon ATG7
Published in OncoImmunology, 2020
Michael D. Arensman, Xiaoran S. Yang, Wenyan Zhong, Stephanie Bisulco, Erik Upeslacis, Edward C. Rosfjord, Shibing Deng, Robert T. Abraham, Christina H. Eng
Immunohistochemistry and digital image analysis were performed as previously described.48 Excised tumors were fixed in 10% neutral buffered formalin for 48 hours prior to being paraffin embedded. Five micron sections were deparaffinized in xylene and rehydrated through a graded series of alcohols to deionized water. Sections underwent heat-induced epitope retrieval in Borg Decloaker (Biocare Medical) for 30 minutes, endogenous peroxidase block with Peroxidazed 1 (Biocare Medical) for 10 min, and protein block with Background Punisher (Biocare Medical) for 10 min. Anti-CD3 (Clone SP162, Abcam #ab135372) or anti-CD8 (Clone D4W2Z, Cell Signaling #98941) was applied at 1:300 for 60 minutes followed by MACH2 Rabbit HRP-Polymer (Biocare Medical) for 30 minutes, and Vina Green chromogen (Biocare Medical) for 12 minutes. After chromogen staining, slides were rinsed in dH2O, counterstained for 10 seconds in Tacha’s hematoxylin (Biocare Medical), dehydrated in 100% alcohol, cleared in xylene, and coverslipped with Permount medium (Fisher). When dry, slides were scanned on a Leica/Aperio AT2 whole slide digital scanner and analyzed using custom algorithms created in Visiopharm software. Visiopharm IHC marker applications with threshold parameters were applied uniformly to identify CD3 and CD8 chromogen-positive area in viable regions of tumor sections.