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![Schematic representation of green fluorescent protein (GFP)-fluorescence emission in a eukaryotic cell: In situ environment, the cellular spread of GFP-emitting fluorescence (left). GFP under chemical fixation with paraformaldehyde (PFA); in some PFA-GFP complexes, a conformational change of GFP is induced, and as consequence GFP remains non-fluorescent (right). Based on Pereira et al. [47].](publication/358133281/figure/fig3/AS:1122928196354049@1644738353517/Schematic-representation-of-green-fluorescent-protein-GFP-fluorescence-emission-in-a.png)
Schematic representation of green fluorescent protein (GFP)-fluorescence emission in a eukaryotic cell: In situ environment, the cellular spread of GFP-emitting fluorescence (left). GFP under chemical fixation with paraformaldehyde (PFA); in some PFA-GFP complexes, a conformational change of GFP is induced, and as consequence GFP remains non-fluorescent (right). Based on Pereira et al. [47].
Source publication
Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a meth...
Contexts in source publication
Context 1
... in the structural integrity of fluorescent proteins may compromise its emission of fluorescence; GFP requires strong fixation to preserve its conformation; especially when GFP is located deeply within the tissue a long-term fixation is required. However, it produces autofluorescence, protein crosslinking, and epitope masking, leading to protein mislocalization due to the absence of fluorescence ( Figure 6) [47,48]. The authors used their improved protocol by applying microwaves for the unmasking of epitopes, and 1% Triton X-100 for permeabilization to visualize deep intracellular and cell-surface GFP signals in human, rat, and mouse heart samples. ...
Context 2
... is located deeply within the tissue a long-term fixation is required. However, it produces autofluorescence, protein crosslinking, and epitope masking, leading to protein mislocalization due to the absence of fluorescence (Figure 6) [47,48]. The authors used their improved protocol by applying microwaves for the unmasking of epitopes, and 1% Triton X-100 for permeabilization to visualize deep intracellular and cell-surface GFP signals in human, rat, and mouse heart samples. ...
Citations
... Research in biology and pathology uses this methodology [16]. Figure 1 presents the basic processes of immunofluorescence as described by Piña et al. [17]. ...
Fish health management is critical to aquaculture and fisheries as it directly affects sustainability and productivity. Fish disease diagnosis has taken a massive stride because of advances in immunological and molecular diagnostic tools which provide a sensitive, quick, and accurate means of identifying diseases. This review presents an overview of the main molecular and immunological diagnostic methods for determining the health of fish. The immunological techniques help to diagnose different fish diseases by detecting specific antigens and antibodies. The application of immunological techniques to vaccine development is also examined in this review. The genetic identification of pathogens is made possible by molecular diagnostic techniques that enable the precise identification of bacterial, viral, and parasitic organisms in addition to evaluating host reactions and genetic variation associated with resistance to disease. The combination of molecular and immunological methods has resulted in the creation of novel techniques for thorough evaluation of fish health. These developments improve treatment measures, pathogen identification and provide new information about the variables affecting fish health, such as genetic predispositions and environmental stresses. In the framework of sustainable fish farming and fisheries management, this paper focuses on the importance of these diagnostic techniques that play a crucial role in protecting fish populations and the aquatic habitats. This review also examines the present and potential future directions in immunological and molecular diagnostic techniques in fish health.
Graphical Abstract
... for details of primary and secondary antibodies). Additionally, the secretory function of the endometrial glandlike structures was assessed by staining for MUC1 (sc-7313, Santa Cruz Biotechnology), with a protocol adapted from Piña et al. 31 . The details are described in the Supplementary Information. ...
To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.
... Immunofluorescence assay (IFA) is a technique in which a fluorescent antibody or antigen is used as a probe to detect an unknown antigen or antibody (Piña et al., 2022). Bedeković et al. developed an IFA method for the diagnosis of BVDV in which a specific monoclonal antibody for BVDV was used as primary antibody and an anti-mouse antibody conjugated with fluorescein isothiocyanate (FITC) as secondary antibody. ...
Bovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea (BVD), which results in significant economic losses in the global cattle industry. Fortunately, various diagnostic methods available for BVDV have been established. They include etiological methods, such as virus isolation (VI); serological methods, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and immunohistochemistry (IHC); molecular methods, such as reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, digital droplet PCR (ddPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR-Cas system; and biosensors. This review summarizes the current diagnostic methods for BVDV, discussing their advantages and disadvantages, and proposes future perspectives for the diagnosis of BVDV, with the intention of providing valuable guidance for effective diagnosis and control of BVD disease.
... Fluorescent markers that use antibodies bound to fluorochromes are among the most utilized. They enable both quantitative and qualitative analyses of protein expression in various cell types (Mortensen and Larsson, 2001;McKinnon, 2018;Piña et al., 2022). The specificity and sensitivity of these fluorescent markers allow for the determination of the concentration and quantity of target molecules through the analysis of relative fluorescence intensity between control and experimental groups in quantitative approaches (Majumder and Fisk, 2014;Ng et al., 2018;Cheung et al., 2021). ...
Fluorescence is used in various biological assays due to its high sensitivity, versatility, and precision. In recent years, studies using medicinal plant extracts have increased. However, fluorescence-based assays could be biased by plant metabolites autofluorescence. To address this issue, this study investigated the interference caused by methanolic extracts and chloroform fractions of three medicinal plants in three fluorescence-based assays on gastric cancer stem cells(CSC): resazurin reduction, confocal microscopy, and flow cytometry. CSC were isolated based on CD44 surface marker, incubated with methanolic extracts and chloroform fractions of Buddleja incana, Dracontium spruceanum, Piper aduncum. Resazurin assay evidenced that CSC exposed to extracts and fractions from the three plants showed significant differences in relative fluorescence units (RFU) levels (p < 0.001) compared to the unexposed groups after a 3-hour incubation. In addition, DMSO-treated CSC exposed to extracts and fractions had significantly lower fluorescence levels than living ones, but higher than extracts and fractions without cells. In confocal microscopy, cancer stem cells exposed to extracts and fractions of B. incana and P. aduncum were observed in the same emission spectra of the CSC markers. In flow cytometry, CSC exposed to extracts and fractions without any fluorescent dyes were detected in the double positive quadrants for CSC markers (CD44+/CD133 + ). Among the three plants, D. spruceanum exhibited the least interference. These results show that methanolic extracts and chloroform fractions contain autofluorescent metabolites that interfere with fluorescence-based assays. These results highlight the importance of a prior evaluation for possible fluorescence interference to avoid interpretation biases in fluorescence assays.
... Subsequently, treatments were carried out, and then the cells were fixed at room temperature with cold acetone for 15 min under agitation. Cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min and then blocked with 2% BSA [58]. Antibodies against 8-OHdG (1:500), lamin B1 (1:1000), PCNA (1:1000), and Klotho (1:1000) were added and incubated overnight at 4 • C. Cells were then incubated for 2 h with secondary antibodies Alexa Fluor TM 488 and Alexa Fluor TM 594 at room temperature in the dark. ...
D-galactose has been widely used as an inducer of cellular senescence and pathophysiological processes related to aging because it induces oxidative stress. On the other hand, the consumption of antioxidants such as curcumin can be an effective strategy to prevent phenotypes related to the enhanced production of reactive oxygen species (ROS), such as aging and senescence. This study aimed to evaluate the potential protective effect of curcumin on senescence and oxidative stress and endoplasmic reticulum stress induced by D-galactose treatment in Lilly Laboratories Culture-Porcine Kidney 1 (LLC-PK1) and human kidney 2 (HK-2) proximal tubule cell lines from pig and human, respectively. For senescence induction, cells were treated with 300 mM D-galactose for 120 h and, to evaluate the protective effect of the antioxidant, cells were treated with 5 µM curcumin for 24 h and subsequently treated with curcumin + D-galactose for 120 h. In LLC-PK1 cells, curcumin treatment decreased by 20% the number of cells positive for senescence-associated (SA)-β-D-galactosidase staining and by 25% the expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and increased by 40% lamin B1 expression. In HK-2 cells, curcumin treatment increased by 60% the expression of proliferating cell nuclear antigen (PCNA, 50% Klotho levels, and 175% catalase activity. In both cell lines, this antioxidant decreased the production of ROS (20% decrease for LLC-PK1 and 10 to 20% for HK-2). These data suggest that curcumin treatment has a moderate protective effect on D-galactose-induced senescence in LLC-PK1 and HK-2 cells.
... For the Wolf group, however, a uniform blue signal was noted on DAPI staining. This might be due to autofluorescene of the samples after treatment with Triton X-100 [28]. The short rinse with PBS at the end of this method might not have been sufficient to entirely wash away residual detergent, resulting in a strong fluorescent signal. ...
BACKGROUND:
Decellularized matrices are an attractive choice of scaffold in regenerative medicine as they can provide the necessary extracellular matrix (ECM) components, signals and mechanical properties. Various detergent-based protocols have already been proposed for decellularization of skeletal muscle tissue. However, a proper comparison is difficult due to differences in species, muscle origin and sample sizes. Moreover, a thorough evaluation of the remaining acellular matrix is often lacking.
METHODS:
We compared an in-house developed decellularization protocol to four previously published methods in a standardized manner. Porcine skeletal muscle samples with uniform thickness were subjected to in-depth histological, ultrastructural, biochemical and biomechanical analysis. In addition, 2D and 3D cytocompatibility experiments were performed.
RESULTS:
We found that the decellularization methods had a differential effect on the properties of the resulting acellular matrices. Sodium deoxycholate combined with DNase I was not an effective method for decellularizing thick skeletal muscle tissue. Triton X-100 in combination with trypsin, on the other hand, removed nuclear material but not cytoplasmic proteins at low concentrations. Moreover, it led to significant alterations in the biomechanical properties. Finally, sodium dodecyl sulphate (SDS) seemed most promising, resulting in a drastic decrease in DNA content without major effects on the ECM composition and biomechanical properties. Moreover, cell attachment and metabolic activity were also found to be the highest on samples decellularized with SDS.
CONCLUSIONS:
Through a newly proposed standardized analysis, we provide a comprehensive understanding of the impact of different decellularizing agents on the structure and composition of skeletal muscle. Evaluation of nuclear content as well as ECM composition, biomechanical properties and cell growth are important parameters to assess. SDS comes forward as a detergent with the best balance between all measured parameters and holds the most promise for decellularization of skeletal muscle tissue.
... Cells were grown in a slide chamber and treated with shControl, shSIRT7, DDX3X/WT, and DDX3X/55Q plasmids along with sorafenib, as described above. Treated cells were washed with PBS following a previously reported protocol (Pina et al., 2022). Cells were incubated with anti-G3BP1, anti-NLRP3, and anti-ASC antibodies and Alexa Fluor 488-conjugated mouse or Alexa Fluor 568-conjugated rabbit antibodies. ...
Aims
Sirtuin 7 (SIRT7) plays an important role in tumor development, and has been characterized as a potent regulator of cellular stress. However, the effect of SIRT7 on sorafenib acquired resistance remains unclear and a possible anti-tumor mechanism beyond this process in HCC has not been clarified. We examined the therapeutic potential of SIRT7 and determined whether it functions synergistically with sorafenib to overcome chemoresistance.
Methods
Cancer Genome Atlas-liver HCC data and unbiased gene set enrichment analyses were used to identify SIRT7 as a potential effector molecule in sorafenib acquired resistance. Two types of SIRT7 chemical inhibitors were developed to evaluate its therapeutic properties when synergized with sorafenib. Mass spectrometry was performed to discover a direct target of SIRT7, DDX3X, and DDX3X deacetylation levels and protein stability were explored. Moreover, an in vivo xenograft model was used to confirm anti-tumor effect of SIRT7 and DDX3X chemical inhibitors combined with sorafenib.
Results
SIRT7 inhibition mediated DDX3X depletion can re-sensitize acquired sorafenib resistance by disrupting NLRP3 inflammasome assembly, finally suppressing hyperactive ERK1/2 signaling in response to NLRP3 inflammasome-mediated IL-1β inhibition.
Conclusions
SIRT7 is responsible for sorafenib acquired resistance, and its inhibition would be beneficial when combined with sorafenib by suppressing hyperactive pro-cell survival ERK1/2 signaling.
... For identi cation of endometrial gland-like structures, a primary antibody against FOXA2 was used (see Table 2 for details of primary and secondary antibody). Additionally, the secretory function of the endometrial gland-like structures was assessed by staining for MUC1 (sc-7313, Santa Cruz Biotechnology), with a protocol adapted from Piña et al. (2022) 29 . The details are described in the supplementary information. ...
... For identi cation of endometrial gland-like structures, a primary antibody against FOXA2 was used (see Table 2 for details of primary and secondary antibody). Additionally, the secretory function of the endometrial gland-like structures was assessed by staining for MUC1 (sc-7313, Santa Cruz Biotechnology), with a protocol adapted from Piña et al. (2022) 29 . The details are described in the supplementary information. ...
To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 ( Muc1 ) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 ( IL6 ) and prostaglandin F synthase ( PGFS ) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.
... ; https://doi.org/10.1101/2024.01.10.575076 doi: bioRxiv preprint IF techniques were improved by utilizing antibody signaling enhancing buffers to increase the permeabilization of cells (Piña et al., 2022) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made ...
Neuropsychiatric disorders that result from stress exposure, including post-traumatic stress disorder and substance abuse, are highly associated with central inflammation. Our previous work established that females selectively exhibit increased proinflammatory cytokine release within the noradrenergic locus coeruleus (LC) in response to witnessing social stress, which was associated with a hypervigilant phenotype. Thus, neuroimmune activation in the LC may be responsible for the heightened risk of developing mental health disorders following stress in females. Further, ablation of microglia using pharmacological techniques reduces the hypervigilant behavioral response exhibited by females during social stress. Therefore, these studies were designed to further investigate the impact of stress-induced neuroimmune signaling on the long-term behavioral and neuronal consequences of social stress exposure in females using DREADDs. We first characterized the use of an AAV-CD68-Gi-DREADD virus targeted to microglia within the LC. While the use of AAVs in preclinical research has been limited by observations regarding poor transfection efficiency in mice, recent data suggest that species specific differences in microglial genetics may render rats more receptive to chemogenetic targeting of microglia using a CD68 promoter. Therefore, clozapine-n-oxide (CNO) was used to activate the microglial expressed hM4Di to inhibit microglial activity during acute exposure to vicarious social defeat (witness stress, WS) in female rats. Neuroimmune activity within the LC, quantified by microglial morphology and cytokine release, was augmented by WS and prevented by chemogenetic microglial inhibition. Following confirmation of DREADD selectivity and efficacy, we utilized this technique to inhibit microglial activity during repeated WS. Subsequently, rats were tested in a marble burying paradigm and exposed to the WS cues and context to measure hypervigilant behaviors. Chemogenetic-mediated inhibition of microglial activity prior to each WS exposure prevented both acute and long-term hypervigilant responses induced by WS across multiple behavioral paradigms. Further, a history of microglial inactivation during WS prevented the heightened LC activity typically observed in response to stress cues. These studies are among the first to use a chemogenetic approach to inhibit microglia within the female brain in vivo and establish LC inflammation as a key mechanism underlying the behavioral and neuronal responses to social stress in females.
... However, the penetration of molecular labels into deep-tissue areas is challenging owing to the incomplete permeability of whole brain tissues [77]. Antibody penetration and binding of specific epitopes for fluorescent immunolabeling rely on a tissue permeabilization step using detergents or enzymes [78,79]. Poor permeabilization can result in loss of cellular mass, including proteins [80] and tissue distortion [81]. ...
Optical brain clearing combined with immunolabeling is valuable for analyzing molecular tissue structures, including complex synaptic connectivity. However, the presence of aberrant lipid deposition due to aging and brain disorders poses a challenge for achieving antibody penetration throughout the entire brain volume. Herein, we present an efficient brain-wide immunolabeling method, the immuno-active clearing technique (iACT). The treatment of brain tissues with a zwitterionic detergent, specifically SB3-12, significantly enhanced tissue permeability by effectively mitigating lipid barriers. Notably, Quadrol treatment further refines the methodology by effectively eliminating residual detergents from cleared brain tissues, subsequently amplifying volumetric fluorescence signals. Employing iACT, we uncover disrupted axonal projections within the mesolimbic dopaminergic (DA) circuits in 5xFAD mice. Subsequent characterization of DA neural circuits in 5xFAD mice revealed proximal axonal swelling and misrouting of distal axonal compartments in proximity to amyloid-beta plaques. Importantly, these structural anomalies in DA axons correlate with a marked reduction in DA release within the nucleus accumbens. Collectively, our findings highlight the efficacy of optical volumetric imaging with iACT in resolving intricate structural alterations in deep brain neural circuits. Furthermore, we unveil the compromised integrity of DA pathways, contributing to the underlying neuropathology of Alzheimer’s disease. The iACT technique thus holds significant promise as a valuable asset for advancing our understanding of complex neurodegenerative disorders and may pave the way for targeted therapeutic interventions.
Graphical Abstract
The axonal projection of DA neurons in the septum and the NAc showed dystrophic phenotypes such as growth cone-like enlargement of the axonal terminus and aggregated neurites. Brain-wide imaging of structural defects in the neural circuits was facilitated with brain clearing and antibody penetration assisted with SB3-12 and Quadrol pre-treatment. The whole volumetric imaging process could be completed in a week with the robust iACT method. Created with https://www.biorender.com/ .
