Fluorescent Immunohistochemistry

2021 ◽  
pp. 131-146
Author(s):  
Vanessa Moreno ◽  
Elizabeth A. Smith ◽  
Sergio Piña-Oviedo
Keyword(s):  
Fluorescent Immunohistochemistry

scholarly journals Characterization of Oral Squamous Cell Carcinoma Associated Inflammation: A Pilot Study

2021 ◽  
Vol 2 ◽  
Author(s):  
Catherine Laliberté ◽  
Nicole Ng ◽  
Denise Eymael ◽  
Kevin Higgins ◽  
Aiman Ali ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Immune Cells ◽  
Tumor Development ◽  
Tissue Analysis ◽  
Fluorescent Immunohistochemistry ◽  
Pro Inflammatory Cytokines

Background: Oral squamous cell carcinoma (OSCC) is a devastating disease that is usually associated with a dense associated inflammatory infiltrate. Characterizing tumor-associated inflammation is critical to understand the pathogenies of tumor development and progression.Methods: We have tested a protocol to analyze tissue and salivary immune cells and mediators of 37 patients with OSCC at different stages and compared to eight chronic periodontitis patients and 24 healthy controls. Tissue analysis was based on fluorescent immunohistochemistry (FIHC) and inflammatory mediators were analyzed using a Luminex-based 30-Plex panel. Immune cells were analyzed using multichannel flow cytometry including CD45, CD66b, CD3, CD4, CD8, CD25, CD56, CD68, CD138, PD-1, and PD-L1.Results: We show an increase in OSCC-associated inflammation characterized by increased pro-inflammatory cytokines including IL-6, IL-8, TNFα, and GMCSF and increased salivary immune cells.Conclusion: We described a new method to analyze salivary inflammatory markers that can be used in future studies to monitor disease progression and prognosis.

A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry

2005 ◽  
Vol 15 (1) ◽  
pp. 21-29 ◽  
Author(s):  
Paul D. Smith ◽  
Karen J. McLean ◽  
Michael A. Murphy ◽  
Yvette Wilson ◽  
Mark Murphy ◽  
...  
Keyword(s):  
Quantitative Assessment ◽  
Fluorescent Immunohistochemistry ◽  
Area Product

scholarly journals Automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification using antibodies from the same species

2015 ◽  
Vol 3 (Suppl 2) ◽  
pp. P111 ◽  
Author(s):  
Wenjun Zhang ◽  
Antony Hubbard ◽  
Tobin Jones ◽  
Srabani Bhaumik ◽  
Adriana Racolta ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Tyramide Signal Amplification ◽  
Fluorescent Immunohistochemistry

scholarly journals Leptin and the Control of Food Intake: Neurons in the Nucleus of the Solitary Tract Are Activated by Both Gastric Distension and Leptin

Endocrinology ◽  
2007 ◽  
Vol 148 (5) ◽  
pp. 2189-2197 ◽  
Author(s):  
Lihong Huo ◽  
Lisa Maeng ◽  
Christian Bjørbæk ◽  
Harvey J. Grill
Keyword(s):  
Food Intake ◽  
Nucleus Tractus Solitarius ◽  
Area Postrema ◽  
Significant Proportion ◽  
Gastric Distension ◽  
Reduce Food Intake ◽  
Signal Transducer ◽  
Electrophysiological Response ◽  
Fluorescent Immunohistochemistry ◽  
Transcription 3

Leptin reduces food intake by an unspecified mechanism. Studies show that forebrain ventricular leptin delivery increases the inhibitory effects of gastrointestinal (GI) stimulation on intake and amplifies the electrophysiological response to gastric distension in neurons of the medial subnucleus of the nucleus tractus solitarius (mNTS). However, forebrain ventricular delivery leaves unspecified the neuroanatomical site(s) mediating leptin’s effect on intake. Detailed anatomical analysis in rats and mice by phosphorylated signal transducer and activator of transcription 3 immunohistochemistry shows that hindbrain leptin-responsive neurons are located exclusively within the mNTS. Here, we investigate 1) whether leptin and gastric distension affect the same mNTS neurons and 2) whether the intake-inhibitory action of gastric distension is potentiated by hindbrain leptin delivery. Twenty-five minutes after gastric balloon distension or sham distension, rats were injected with leptin or vehicle and killed 35 min later. Double-fluorescent immunohistochemistry for phosphorylated signal transducer and activator of transcription 3 and c-Fos revealed that about 40% of leptin-responsive cells also respond to gastric distension. A paradigm was then developed to examine the relationship between leptin and gastric distension volume on intake inhibition. At subthreshold levels, hindbrain ventricular leptin or distension volume were without effect. When combined, an interaction occurred that significantly reduced food intake. We conclude that 1) leptin-responsive neurons in the hindbrain are primarily located in the mNTS at the level of the area postrema, a key vagal afferent projection zone of the GI system; 2) a significant proportion of leptin-responsive neurons in the mNTS are activated by stomach distension; and 3) leptin delivered to the hindbrain is sufficient to potentiate the intake-suppressive effects of an otherwise ineffective volume of gastric distension. These results are consistent with the hypothesis that leptin acts directly on neurons within the mNTS to reduce food intake through an interaction with GI signal processing.

scholarly journals Correlated Sensory and Sympathetic Innervation Between the Acupoint BL23 and Kidney in the Rat

2021 ◽  
Vol 14 ◽  
Author(s):  
Zhiyun Zhang ◽  
Dongsheng Xu ◽  
Jia Wang ◽  
Jingjing Cui ◽  
Shuang Wu ◽  
...  
Keyword(s):  
Sympathetic Innervation ◽  
Nerve Fibers ◽  
Neural Pathways ◽  
Visceral Organ ◽  
Target Organs ◽  
Calcitonin Gene ◽  
Neural Tracing ◽  
Cholera Toxin Subunit B ◽  
Spinal Segments ◽  
Fluorescent Immunohistochemistry

Objective: To investigate the sensory and sympathetic innervations associated with both acupoint “Shenshu” (BL23) and kidney in the rat for insight into the neuronal correlation between the Back-Shu Point and its corresponding visceral organ.Methods: The BL23 and kidney were selected as the representative acupoint and visceral organ in this study, in which their local nerve fibers were examined by using double fluorescent immunohistochemistry with calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH). Meanwhile, their neuronal correlation in the dorsal root ganglia (DRGs), spinal cord, and sympathetic (paravertebral) chain were investigated using a double fluorescent neural tracing technique with Alexa Fluor 488 and 594 conjugates with cholera toxin subunit B (AF488/594-CTB).Results: The local tissue of acupoint BL23 and the fibrous capsule of kidney distributed abundantly with CGRP- and TH-positive nerve fibers, corresponding to their sensory and sympathetic innervation. On the other hand, the sensory neurons associated with acupoint BL23 and kidney were labeled with AF488/594-CTB and distributed from thoracic (T) 11 to lumbar (L) 3 DRGs and from T10 to L2 DRGs, respectively, in which some of them in T12-T13 DRGs were simultaneously labeled with both AF488/594-CTB. Also, postganglionic neurons associated with both acupoint BL23 and kidney were found in the sympathetic chain at the same spinal segments but separately labeled with AF488-CTB and AF594-CTB.Conclusion: Our study demonstrates the neural characteristics of the acupoint BL23 and kidney in the rat from the perspective of neurochemistry and neural pathways, providing an example for understanding the neuronal correlation between the Back-Shu Points and their corresponding visceral organs. These results suggest that the stimulation of the Back-Shu Points may regulate the activities of the target-organs via the periphery sensory and sympathetic pathways.

scholarly journals Nr5a1b promotes and Nr5a2 inhibits transcription of lhb in the orange-spotted grouper, Epinephelus coioides†

2019 ◽  
Vol 101 (4) ◽  
pp. 800-812
Author(s):  
Boyang Shi ◽  
Huijie Lu ◽  
Lihong Zhang ◽  
Weimin Zhang
Keyword(s):  
Differential Expression ◽  
Chromatin Immunoprecipitation ◽  
Regulatory Mechanisms ◽  
Epinephelus Coioides ◽  
Antagonistic Effects ◽  
Opposite Trend ◽  
Vitellogenic Stage ◽  
Lh Cells ◽  
Fluorescent Immunohistochemistry

Abstract Nr5a1 (Sf-1) up-regulates lhb expression across vertebrates; however, its regulatory roles on fshb remain to be defined. Moreover, the involvement of Nr5a2 in the regulation of gonadotropin expression is not clear either. In the present study, the involvement of Nr5a1b (a homologue of Nr5a1) and Nr5a2 in the regulation of lhb and fshb expression in the orange-spotted grouper was examined. Dual fluorescent immunohistochemistry using homologous antisera showed that in the pituitary of orange-spotted groupers, Lh cells contain both immunoreactive Nr5a1b and Nr5a2 signals, whereas Fsh cells contain neither of them. In LβT2 cells, Nr5a1b up-regulated basal activities of lhb and fshb promoters possibly via Nr5a sites, and synergistically (on lhb promoter) or additively (on fshb promoter) with forskolin. Surprisingly, Nr5a2 inhibited basal activities of lhb promoter possibly via Nr5a sites and attenuated the stimulatory effects of both forskolin and Nr5a1b. In contrast, Nr5a2 had no effects on fshb promoter. Chromatin immunoprecipitation analysis showed that both Nr5a1b and Nr5a2 bound to lhb promoter, but not fshb promoter in the pituitary of the orange-spotted grouper. The abundance of Nr5a1b bound to lhb promoter was significantly higher at the vitellogenic stage than the pre-vitellogenic stage, whereas that of Nr5a2 exhibited an opposite trend. Taken together, data of the present study demonstrated antagonistic effects of Nr5a1b and Nr5a2 on lhb transcription in the orange-spotted grouper and revealed novel regulatory mechanisms of differential expression of lhb and fshb genes through Nr5a homologues in vertebrates.

scholarly journals Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies

2017 ◽  
Vol 97 (7) ◽  
pp. 873-885 ◽  
Author(s):  
Wenjun Zhang ◽  
Antony Hubbard ◽  
Tobin Jones ◽  
Adriana Racolta ◽  
Srabani Bhaumik ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Tyramide Signal Amplification ◽  
Fluorescent Immunohistochemistry

scholarly journals P03.26 Immunoprofiling of oral and oropharyngeal tumors of different etiology

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A33.1-A33
Author(s):  
B Pokrývková ◽  
M Grega ◽  
J Klozar ◽  
O Vencalek ◽  
J Nunvar ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Immune Cells ◽  
Demographic Data ◽  
Tumor Infiltrating Lymphocytes ◽  
Tumor Parenchyma ◽  
Fluorescent Immunohistochemistry ◽  
Infiltrating Lymphocytes ◽  
Formalin Fixed ◽  
Selection Of

BackgroundHead and neck carcinomas (HNC) are the world’s sixth most common cancer. Most of HNCs are associated with tobacco and other environmental factors but a growing part of oropharyngeal tumors are caused by persistent infection of human papillomavirus (HPV). Patients with HPV positive cancers have a better prognosis with fewer recurrences. This may be caused by different anti-tumor immune response and immune profile of patients. Multispectral fluorescent immunohistochemistry (fIHC) is a powerful tool for a detailed analysis of the tumor microenvironment. This method allows to access the phenotype and calculate cells in tumor parenchyma and stroma of the tumor since in comparison to flow cytometry, an architecture of the tissue remains preserved. fIHC is uniquely suited to study interaction of immune and cancer cells in situ.Materials and MethodsNumber of 97 formalin fixed paraffine-embedded slides of the human HNC tissue with known etiology were examined using 4 different panels of 5 antibodies each. These panels include antibodies suitable for phenotyping of immune cells (CD3e, CD4, CD8, FOXP3) or their functional description (PD1, CTLA4, ICOS, CCR4). Additionally, antibodies against Ki67, VEGF and cell cytokeratin were used. Slides were stained using Opal™ 7-Color Fluorescent IHC Kit (Akoya Biosciences). The quantity of immune cells was evaluated in stroma and tumor compartment using InForm™ 2.4.6. software (Akoya Biosciences). For all patients the demographic and clinical data were available and these patients were followed for up to 18 years.ResultsOur results have shown significantly higher abundance of Th and Tc in both compartments of HPV+ samples. Besides HPV etiology Th and Tc in the tumor microenvironment predict independently better survival of patients. We did not observed difference in number of Tregs (characterized as a CD3+CD4+FOXP3+ cells) in tumors of different etiology, but we detected higher number of ICOS+Tregs in stroma of HPV- tumors. We also quantified the subpopulations of Th and Tc cells expressing regulatory receptors PD1 and CTLA4. PD1 showed significantly higher expression on Th and Tc both in tumor and stroma of HPV+ tumors, but CTLA4 expression was significantly higher only on Th located in stroma of HPV- tumors. Moreover, we detected significantly higher VEGF expression in both compartments and higher proliferating activity of tumor cells in HPV- tumors.ConclusionsDetailed analyses of the tumor infiltrating lymphocytes allows for selection of prognostic markers in HNC of different etiology. Our results may also help to understand the better prognosis of HPV+ patients. More detailed survival analyses with inclusion of other clinical and demographic data will be presented.Disclosure InformationB. Pokrývková: None. M. Grega: None. J. Klozar: None. O. Vencalek: None. J. Nunvar: None. R. Tachezy: None.

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