Browsing by Subject "Immunofluorescence"
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Item A guide to selecting high-performing antibodies for PLC-gamma-2 for use in Western Blot, immunoprecipitation and immunofluorescence(Taylor & Francis, 2024-01-18) Ruíz Moleón, Vera; Fotouhi, Maryam; Alende, Charles; Ayoubi, Riham; Bedford, Logan M.; Southern, Kathleen; Richardson, Timothy I.; Laflamme, Carl; NeuroSGC/YCharOS/EDDU collaborative group; ABIF consortium; Pharmacology and Toxicology, School of MedicinePhosphatidylinositol-specific phospholipase C gamma 2 (PLC-gamma-2) is an enzyme that regulates the function of immune cells. PLC-gamma-2 has been implicated in neurodegenerative and autoimmune disorders, yet investigation of this protein has been limited by a lack of independently characterized antibodies. Here we have characterized eleven PLC-gamma-2 commercial antibodies for use in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.Item Coffee Intake of Colorectal Cancer Patients and Prognosis According to Histopathologic Lymphocytic Reaction and T-Cell Infiltrates(Elsevier, 2022) Ugai, Tomotaka; Haruki, Koichiro; Väyrynen, Juha P.; Borowsky, Jennifer; Fujiyoshi, Kenji; Lau, Mai Chan; Akimoto, Naohiko; Zhong, Rong; Kishikawa, Junko; Arima, Kota; Shi, Shan-shan; Zhao, Melissa; Fuchs, Charles S.; Zhang, Xuehong; Giannakis, Marios; Song, Mingyang; Nan, Hongmei; Meyerhardt, Jeffrey A.; Wang, Molin; Nowak, Jonathan A.; Ogino, Shuji; Community and Global Health, Richard M. Fairbanks School of Public HealthGiven previous biological evidence of immunomodulatory effects of coffee, we hypothesized that the association between coffee intake of colorectal cancer patients and survival differs by immune responses. Using a molecular pathological epidemiology database of 4,465 incident colorectal cancer cases, including 1,262 cases with molecular data, in the Nurses’ Health Study and the Health Professionals Follow-up Study, we examined the association between coffee intake of colorectal cancer patients and survival in strata of levels of histopathologic lymphocytic reaction and T-cell infiltrates in tumor tissue. We did not observe a significant association of coffee intake with colorectal cancer-specific mortality [multivariable-adjusted hazard ratio (HR) for one cup increase of coffee intake per day, 0.93; 95% confidence interval (CI), 0.84-1.03]. Although statistical significance was not reached at the stringent level (α=0.005), the association of coffee intake with colorectal cancer-specific mortality differed by Crohn's-like lymphoid reaction (Pinteraction=.007). Coffee intake was associated with lower colorectal cancer-specific mortality in patients with high Crohn's-like reaction (multivariable HR for one cup increase of coffee intake per day, 0.55; 95% CI, 0.37–0.81; Ptrend=.002), but not in patients with intermediate Crohn's-like reaction (the corresponding HR, 1.02; 95% CI, 0.72–1.44) or negative/low Crohn's-like reaction (the corresponding HR, 0.95; 95% CI, 0.83–1.07). The associations of coffee intake with colorectal cancer-specific mortality did not significantly differ by levels of other lymphocytic reaction or any T-cell subset (Pinteraction>.18). There is suggestive evidence for differential prognostic effects of coffee intake by Crohn’s-like lymphoid reaction in colorectal cancer.Item Neuromuscular junction visualization in paraffin‐embedded thyroarytenoid muscle sections: Expanding options beyond frozen section analysis(Wiley, 2025-01-07) Kaefer, Samuel L.; Shay, Elizabeth O.; Morrison, Rachel A.; Zhang, Lujuan; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of MedicineObjectives: The current gold standard for immunofluorescent (IF) visualization of neuromuscular junctions (NMJs) in muscle utilizes frozen tissue sections with fluorescent conjugated antibodies to demarcate neurons and IF alpha-bungarotoxin (α-BTX) to demarcate motor endplates. Frozen tissue sectioning comes with inherent inescapable limitations, including cryosectioning artifact and limited sample shelf-life. However, a parallel approach to identify NMJs in paraffin-embedded tissue sections has not been previously described. Methods: Yucatan minipig thyroarytenoid (TA) muscle was harvested and prepared as 5-μm thick paraffin-embedded tissue sections. A variety of antibodies at various concentrations were selected to target nicotinic acetylcholine receptors, synaptic vesicles, and neurons. Results: Neurofilament (NEFL, Invitrogen, 1:500) and synaptic vesicle glycoprotein (SV2, DSHB, 1:230) bound and demarcated the neurons and synaptic vesicles, respectively. Following consistent visualization of nerve tissue, rabbit anti-nicotinic acetylcholine receptor alpha-1 subunit (CHRNα1, Abcam, 1:500) was used to identify the acetylcholine receptors within motor endplates. Complete NMJ visualization was then achieved with an optimized protocol using primary antibodies to the neurofilament light chain, nerve synaptic vesicle glycoprotein 2, and the alpha 1 subunit of the nicotinic acetylcholine receptor. Slide imaging was performed with the Echo Revolve Microscope (40×). Conclusions: Herein, we describe a new methodology to visualize NMJs within paraffin-embedded TA muscle sections. Our protocol avoids the known limitations associated with cryosectioned samples and introduces a new neurolaryngologic research tool that utilizes the advantageous ability of paraffin-embedded sectioning to preserve tissue morphology. In conjunction with standard cryosectioned methods, the described paraffin-embedded protocol serves to enhance histological analysis of NMJs.Item Novel method for detection of glycogen in cells(Oxford University Press, 2017-05-01) Skurat, Alexander V.; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.; Biochemistry and Molecular Biology, School of MedicineGlycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing g lutathione S-transferase, a cM c eptitope and the tbd1 BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions.Item Optimizing Confocal Imaging Protocols for Muscle Fiber Typing in the Mouse Masseter Muscle(Bio-protocol, 2025-04-05) Matias, Catalina; Yamada, Chiaki; Movila, Alexandru; Brault, Jeffrey J.; Anatomy, Cell Biology and Physiology, School of MedicineThe masseter muscle, a key orofacial muscle, demonstrates unique anatomical and functional properties, including sexual dimorphism in myosin heavy chain (MyHC) expression and complex fiber architecture. Despite its importance in mastication and relevance to various disorders, phenotypic characterization of the masseter remains limited. Conventional fluorescence microscopy has been a cornerstone in muscle fiber typing, reliably identifying MyHC isoforms and measuring fiber cross-sectional areas. Building on this foundation, confocal microscopy offers complementary advantages, such as enhanced resolution, increased flexibility for multiplexing, and the ability to visualize complex structures in three dimensions. This study presents a detailed protocol for using confocal microscopy to achieve high-resolution imaging and molecular characterization of masseter muscle cryosections. By leveraging advanced technologies such as white light lasers and extended z-length imaging, this method ensures precise spectral separation, simultaneous multichannel fluorescence detection, and the ability to capture muscle architecture in three dimensions. The protocol includes tissue preparation, immunostaining for MyHC isoforms, and postprocessing for fiber segmentation and quantification. The imaging setup was optimized for minimizing signal bleed through, improving the signal-to-noise ratio, and enabling detailed visualization of muscle fibers and molecular markers. Image postprocessing allows for quantification of the cross-sectional area of individual fibers, nuclei location measurements, and identification of MyHC isoforms within each fiber. This confocal microscopy-based protocol provides similar resolution and contrast compared to conventional techniques, enabling robust multiplexed imaging and 3D reconstruction of muscle structures. These advantages make it a valuable tool for studying complex muscle architecture, offering broad applications in muscle physiology and pathology research. Key features • Enables high-resolution imaging of muscle fiber architecture, capturing detailed spatial relationships using extended z-length and advanced spectral separation techniques. • Supports simultaneous detection of multiple molecular markers for robust muscle fiber typing and molecular localization. • Allows for the generation of three-dimensional models to analyze muscle structures such as neuromuscular junctions, extracellular matrix, and mitochondrial organization. • Adaptable to various skeletal muscles and species, providing valuable insights into muscle physiology, regeneration, and disease processes. Graphical overview Analyzing muscle fiber composition and morphology in mice's masseter muscle using confocal microscopy. Workflow for characterizing rodent masseter muscle fibers using advanced confocal microscopy. Confocal microscopy, equipped with white light laser technology and optimized z-stack imaging, allows precise spectral unmixing to reduce bleed through and enhance signal detection. The z-length is extended beyond the physical thickness of the sample to account for potential variations in tissue flatness and ensure complete imaging of all focal planes. The resulting high-resolution images provide detailed insights into fiber architecture, molecular composition, and cross-sectional areas, ensuring robust and reproducible data for analyzing the complex phenotypic characteristics of the masseter and other muscles.Item Purification of SIMPL Antibody and Immunofluorescence of SIMPL Sub-Cellular Localization in Response to TNFα- and IL-1(2011-03-10) Cogill, Steven B.; Harrington, Maureen A.; Goebl, Mark, 1958-; Sanghani, Sonal P.SIMPL is a transcriptional co-activator that alters the activity of transcription factor, NF-κB. In response to pathogens, cytokines such as Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) signal through the IL-1 and TNF-α receptors, respectively, which are found on various cell types. Activation of these receptors can result in the nuclear localization of NF-κB where it enables the transcription of several different genes key in the innate immune response. Endogenous co-localization of the SIMPL protein with NF-κB in response to these same cytokine signals has yet to be demonstrated. Polyclonal antibody generated against a truncated version of the SIMPL protein was purified from the sera obtained from immunized rabbits using affinity chromatography. The antibody was found to have a high specificity for both the native and denatured form of the protein as demonstrated by the lack of nonspecific bands observed in immunoprecipitations and Western blotting. The antibody was utilized in immunofluorescence experiments on mouse endothelial cells that were either unstimulated or were stimulated (IL-1 or TNF-α). In the absence of cytokine, SIMPL was localized in both the cytoplasm and the nucleus as opposed to NF-κB which was almost exclusively localized in the cytoplasm. In the presence of IL-1, the concentration of SIMPL in the nucleus was increased, and in the presence of TNF-α, the concentration of SIMPL in the nucleus was even greater. Results of this study identified future routes for SIMPL antibody isolation as well as to demonstrate that endogenous SIMPL protein nuclear localization may not be solely dependent upon TNF-α signaling.