Immunofluorescence Staining Method Research Articles

Immunofluorescence staining of colorectal cancer patient-derived organoids.

Organoids recapitulate the cellular heterogeneity, functionality, architecture and molecular signature of the organ or diseased tissue from which they are derived. They thus provide a bridge between traditional 2D culture systems and animal models and have profoundly enhanced our ability to study organ development and disease in vitro. Fluorescence microscopy has been an essential method in characterizing the cellular and morphological composition of organoids and demonstrating that they faithfully recapitulate the in vivo tissue of origin. Here we provide a straightforward method for immunofluorescence staining and confocal microscopy imaging of colorectal cancer (CRC) patient-derived organoids (PDOs) in basement matrix. The method is applicable to other types of human organoids, and we have also successfully used it on organoids derived from the mouse mammary gland.

The role of biomarkers in cell counting with U‐Net CNN

AbstractBackgroundImaging methods that are non‐destructive preserve tissue integrity and lead to improved topography analysis for the study of neurodegenerative diseases like Alzheimer’s Disease (AD). Immunofluorescence (IF) staining methods facilitate such investigations by addressing a gradient of intensity variations relevant for in situ cell detection [1] and morphology quantification. This paper reports on the colocalization of proteins and cell structures from human post‐mortem tissue by evaluating IF expression by means of TBR1 and NeuN biomarkers. We compare different U‐Net‐based models by exploring image representations obtained by combining image channels with different biomarkers.MethodFrom an image dataset with 21 full‐size IF micrographs, we augment it by creating a cropping mechanism resulting in 40 to 60 image crops from each micrograph, each crop with 128x128 pixels. Later, we enhance the resulting 1,200 cropped images, and construct 3‐channel (x,y,z) image inputs of dimensions 128x128x3 for the U‐Net model in a two‐fold process: (I) stack all 3 channels with single‐channel IF cropped images corresponding to NeuN biomarkers (NeuN, NeuN, NeuN); (II) stack the single channel images of TBR1, NeuN, and a single image representing the maximum pixel intensities from TBR1 and NeuN images (TBR1, NeuN, max (TBR1, NeuN)). With a test‐train split of 70% ‐ 30%, and parameter tuning, we compare the two models via metrics such as neuron areas, counts, accuracy and recall.ResultThe model trained with channel stacking (II) outperforms the model trained with (I) with an accuracy and recall of 0.96 and 0.5, as opposed to 0.95 and 0.4. We also found a great variation in neuron area and counts as depicted in the images below.ConclusionWhile this is the first step in accelerating the quantification of neurons for the pathophysiology studies necessary for drug development, there is a need in establishing protocols for the generation of datasets with manual segmentation to further train our models. The inclusion of prior information about the expected neuron morphology increases the performance of our networks, and successfully deploy a foreseen automated pipeline. Reference: [1] Ehrenberg et al. A manual multiplex immunofluorescence method for investigating neurodegenerative diseases. Journal of Neuroscience Methods, June, 2020.

Automated Quantitative Image Evaluation of Antigen Retrieval Methods for 17 Antibodies in Placentation and Implantation Diagnostic and Research.

Immunostaining in clinical routine and research highly depends on standardized staining methods and quantitative image analyses. We qualitatively and quantitatively compared antigen retrieval methods (no pretreatment, pretreatment with pepsin, and heat-induced pretreatment with pH 6 or pH 9) for 17 antibodies relevant for placenta and implantation diagnostics and research. Using our newly established, comprehensive automated quantitative image analysis approach, fluorescent signal intensities were evaluated. Automated quantitative image analysis found that 9 out of 17 antibodies needed antigen retrieval to show positive staining. Heat induction proved to be the most efficient form of antigen retrieval. Eight markers stained positive after pepsin digestion, with β-hCG and vWF showing enhanced staining intensities. To avoid the misinterpretation of quantitative image data, the qualitative aspect should always be considered. Results from native placental tissue were compared with sections of a placental invasion model based on thermo-sensitive scaffolds. Immunostaining on placentas in vitro leads to new insights into fetal development and maternal pathophysiological pathways, as pregnant women are justifiably excluded from clinical studies. Thus, there is a clear need for the assessment of reliable immunofluorescent staining and pretreatment methods. Our evaluation offers a powerful tool for antibody and pretreatment selection in placental research providing objective and precise results.

The role of biomarkers in cell counting with U‐Net CNN

AbstractBackgroundImaging methods that are non‐destructive preserve tissue integrity and lead to improved topography analysis for the study of neurodegenerative diseases like Alzheimer’s Disease (AD). Immunofluorescence (IF) staining methods facilitate such investigations by addressing a gradient of intensity variations relevant for in situ cell detection [1] and morphology quantification. This paper reports on the colocalization of proteins and cell structures from human post‐mortem tissue by evaluating IF expression by means of TBR1 and NeuN biomarkers. We compare different U‐Net‐based models by exploring image representations obtained by combining image channels with different biomarkers.MethodFrom an image dataset with 21 full‐size IF micrographs, we perform data augmentation by creating a cropping mechanism resulting in 40 to 60 image crops from each micrograph, each crop with 128x128 pixels. Later, we enhance the resulting 1,200 cropped images, and construct 3‐channel (X,Y,Z) image inputs of dimensions 128x128x3 for the U‐Net model in a two‐fold process: (I) stack all 3 channels with single‐channel IF cropped images corresponding to NeuN biomarkers (NeuN, NeuN, NeuN); (II) stack the single channel images of TBR1, NeuN, and a single image representing the maximum pixel intensities from TBR1 and NeuN images (TBR1, NeuN, max (TBR1, NeuN)). With a test‐train split of 70% ‐ 30%, and parameter tuning, we compare the two models via metrics such as neuron areas, counts, accuracy and recall.ResultThe model trained with channel stacking (II) outperforms the model trained with (I) with an accuracy and recall of 0.96 and 0.5, as opposed to 0.95 and 0.4. We also found a great variation in neuron area and counts as depicted in the images below.ConclusionWhile this is the first step in accelerating the quantification of neurons for the pathophysiology studies necessary for drug development, there is a need in establishing protocols for the generation of datasets with manual segmentation to further train our models. The inclusion of prior information about the expected neuron morphology increases the performance of our networks, and can successfully deploy a foreseen automated pipeline. [1] Ehrenberg et al. A manual multiplex immunofluorescence method for investigating neurodegenerative diseases. Journal of Neuroscience Methods,June, 2020.

Empagliflozin protects diabetic pancreatic tissue from damage by inhibiting the activation of the NLRP3/caspase-1/GSDMD pathway in pancreatic β cells: in vitro and in vivo studies

ABSTRACT Diabetes mellitus is an important public health problem worldwide. Insulin deficiency caused by pancreatic β cell dysfunction is an important pathogenic factor of diabetes mellitus. This study evaluated whether empagliflozin (EMPA) protects the pancreas from diabetes mellitus-induced injury by downregulating the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/caspase-1/Gasdermin D (GSDMD) pyroptosis-related inflammasome pathway in vitro and in vivo. In vivo, animals were separated into blank control (control, C57/bl6j wild-type mice), diabetes model (db/db mice, BKS-Leprem2Cd479/Gpt mice), and db/db mice+EMPA (db/db+EMPA) groups. In vitro, pancreatic β cells were separated into low glucose (control), high glucose (HG), and HG+EMPA groups. The db/db+EMPA group were administered empagliflozin at 10 mg/(kg·day) by gavage for six months. Histological changes in the pancreatic tissues were observed by hematoxylin-eosin staining, and levels of the pyroptosis-related inflammatory factors NLPR3, caspase-1, and GSDMD were measured by immunohistochemistry and immunofluorescence staining methods. The Cell Counting Kit-8 assay was used to detect the effect of different concentrations of glucose and empagliflozin on the proliferation of mouse insulinoma islet β (β TC-6) cells. NLRP3/caspase-1/GSDMD expression was assessed by western blotting and immunofluorescent labeling in the β TC-6 cells. The results showed that empagliflozin reduced the pathological changes and inflammatory cell infiltration in the pancreatic tissues of db/db mice. Furthermore, empagliflozin not only reduced the expression levels of NLRP3/caspase-1/GSDMD in vitro, but also reduced their expression levels in vivo. In summary, our data suggested that empagliflozin protects the pancreatic tissues from diabetes mellitus-induced injury by downregulating the NLRP3/caspase-1/GSDMD pyroptosis-related inflammasome pathway.

Ferruginol Restores SIRT1-PGC-1α-Mediated Mitochondrial Biogenesis and Fatty Acid Oxidation for the Treatment of DOX-Induced Cardiotoxicity.

Background: Doxorubicin (DOX), a broad-spectrum chemotherapy drug, has life-threatening cardiotoxicity. Therefore, searching cardioprotective drugs for DOX-induced cardiotoxicity (DIC) is urgently needed. Objectives: This study aimed to explore cardioprotective effect and specific mechanism by which Ferruginol (FGL) attenuated DIC in vivo and in vitro. Methods: We evaluated the cardioprotection of FGL and performed high-throughput RNA-Seq on a DIC mouse. Whereafter, multiple methods, including western blot, RT-qPCR, a transmission electron microscope, CO-IP, immunofluorescence, and other staining methods, and antagonist of SIRT1 and PGC-1α were utilized to confirm the cardioprotection and molecular mechanism of FGL. Results: FGL-exerted cardioprotection manifested as enhanced cardiac function and reduced structural damage and apoptosis. The transcriptome and other results revealed that FGL facilitated PGC-1α-mediated mitochondrial biogenesis and fatty acid oxidation (MB and FAO) by increasing the expression of PGC-1α and concurrently promoting the expression of SIRT1-enhancing deacetylase SIRT1 deacetylating and activating PGC-1α. Conclusions: These results documented that FGL exerted cardioprotective effects restoring MB&FAO via the SIRT1–PGC-1α axis.

Modification of a Tumor-Targeting Bacteriophage for Potential Diagnostic Applications.

Background: Tumor-targeting bacteriophages can be used as a versatile new platform for the delivery of diagnostic imaging agents and therapeutic cargo. This became possible due to the development of viral capsid modification method. Earlier in our laboratory and using phage display technology, phages to malignant breast cancer cells MDA-MB 231 were obtained. The goal of this study was the optimization of phage modification and the assessment of the effect of the latter on the efficiency of phage particle penetration into MDA-MB 231 cells. Methods: In this work, we used several methods, such as chemical phage modification using FAM-NHS ester, spectrophotometry, phage amplification, sequencing, phage titration, flow cytometry, and confocal microscopy. Results: We performed chemical phage modification using different concentrations of FAM-NHS dye (0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM). It was shown that with an increase of the modification degree, the phage titer decreases. The maximum modification coefficient of the phage envelope with the FAM–NHS dye was observed with 4 mM modifying agent and had approximately 804,2 FAM molecules per phage. Through the immunofluorescence staining and flow cytometry methods, it was shown that the modified bacteriophage retains the ability to internalize into MDA-MB-231 cells. The estimation of the number of phages that could have penetrated into one tumor cell was conducted. Conclusions: Optimizing the conditions for phage modification can be an effective strategy for producing tumor-targeting diagnostic and therapeutic agents, i.e., theranostic drugs.

Strong Twirling-Rotating Manual Acupuncture with 4 r/s Is Superior to 2 r/s in Relieving Pain by Activating C-Fibers in Rat Models of CFA-Induced Pain.

Background Manual acupuncture (MA) with different stimulus frequencies may give rise to varying acupuncture effects. However, the intensity-effect relationship and the underlying mechanisms of MA remain unclear. Objective To compare the analgesic effects of different frequencies of twirling-rotating MA on rats with complete Freund's adjuvant- (CFA-) induced pain and explore the underlying mechanism via peripheral sensory nerves. Methods First, 36 healthy male Wistar rats were randomly divided into 6 groups: control group, 2 r/s MA group (twirling-rotating MA with the frequency of 2 revolutions per second), 4 r/s MA group (twirling-rotating MA with the frequency of 4 revolutions per second), CFA group, CFA + 2 r/s MA group, and CFA + 4 r/s MA group. Rats in three CFA groups received an intraplantar injection of CFA to establish a pain model, while the rats in other three groups received an intraplantar injection of saline. Rats in the 2 r/s MA group and 4 r/s MA group were treated with the corresponding frequencies of twirling-rotating MA on bilateral Zusanli (ST36) and Kunlun (BL60) for 7 days. The ipsilateral nociceptive thresholds (paw withdrawal latency; PWL) were tested to evaluate the analgesic effects. Second, 9 healthy male Wistar rats were randomly divided into 3 groups: control group, 2 r/s MA group, and 4 r/s MA group. The proportion of C-fiber neurons (calcitonin gene-related peptide- (CGRP-) positive neurons) and A-fiber neurons (neurofilament 200- (NF200-) positive neurons) in the dorsal root ganglia (DRG) activated by MA were quantitatively analyzed with the morphological immunofluorescence staining method. Third, 30 healthy male Wistar rats were randomly divided into 6 groups: control group, CFA group, CFA + 2 r/s MA group, CFA + 2 r/s MA + RTX group, CFA + 4 r/s MA group, and CFA + 4 r/s MA + RTX group. Resiniferatoxin (RTX) was injected into the acupoints before acupuncture. PWL was evaluated to investigate the analgesic effect. Results Both types of MA treatment increased the PWL of saline-injecting rats and pain model rats. Moreover, 4 r/s MA was superior to 2 r/s MA in increasing PWL. A higher quantity of excited C-fiber neurons was observed following 4 r/s MA than 2 r/s MA, while the reverse was observed in the activation of A-fiber neurons. Following the injection of RTX to inhibit the activation of C-fibers, the analgesic effect of 4 r/s MA reduced significantly but not of 2 r/s MA. Conclusion Strong MA (4 r/s MA) has superior analgesic effects to gentle MA (2 r/s MA) on CFA model rats, which is associated with C-fiber activation.

MiR-3146 induces neutrophil extracellular traps to aggravate gout flare.

BackgroundGout is an inflammatory arthritis and is characterized by the accumulation of deposited monosodium urate (MSU) crystals in the joints. miRNAs may act as key regulators of gout pathogenesis. The aim of our study was to explore the underlying role and molecular mechanism of miR‐3146 in the formation of neutrophil extracellular traps (NETs) during the pathogenesis of gout.MethodsThe expression of miR‐3146 and sirtuin 1 (SIRT1) was determined by real‐time reverse transcription‐polymerase chain reaction and Western blot, respectively. The luciferase reporter assay was performed to identify the targeting relationship between miR‐3146 and SIRT1. Reactive oxygen species (ROS) production was detected by fluorescent staining. NETs formation was demonstrated via immunofluorescence staining and ELISA method. Gout model was induced in rats to verify the effects of miR‐3146 inhibition on histopathological changes and NETs.ResultsHere, we found miR‐3146 expression was dramatically increased in neutrophils of patients with gout, which was accompanied with the higher levels of NETs. MSU crystals significantly increased miR‐3146 expression and ROS production in neutrophils. The NETs process was also triggered by MSU crystals. Furthermore, we verified the interaction between miR‐3146 and SIRT1. Additionally, antagomir‐3146‐based therapy effectively inhibited the formation of NETs in rats with gout.ConclusionOur findings indicated that miR‐3146‐mediated NETs formation may play a potential role in the pathogenesis of gout. These results suggested that miR‐3146 could be used as a potential therapeutic target for the treatment of gout.

Dl-3-N-Butylphthalide Promotes Angiogenesis in an Optimized Model of Transient Ischemic Attack in C57BL/6 Mice.

Transient ischemic attack (TIA) has been widely regarded as a clinical entity. Even though magnetic resonance imaging (MRI) results of TIA patients are negative, potential neurovascular damage might be present, and may account for long-term cognitive impairment. Animal models that simulate human diseases are essential tools for in-depth study of TIA. Previous studies have clarified that Dl-3-N-butylphthalide (NBP) promotes angiogenesis after stroke. However, the effects of NBP on TIA remain unknown. This study aims to develop an optimized TIA model in C57BL/6 mice to explore the microscopic evidence of ischemic injury after TIA, and investigate the therapeutic effects of NBP on TIA. C57BL/6 mice underwent varying durations (7, 8, 9 or 10 min) of middle cerebral artery occlusion (MCAO). Cerebral artery occlusion and reperfusion were assessed by laser speckle contrast imaging. TIA and ischemic stroke were distinguished by neurological testing and MRI examination at 24 h post-operation. Neuronal apoptosis was examined by TUNEL staining. Images of submicron cerebrovascular networks were obtained via micro-optical sectioning tomography. Subsequently, the mice were randomly assigned to a sham-operated group, a vehicle-treated TIA group or an NBP-treated TIA group. Vascular density was determined by immunofluorescent staining and fluorescein isothiocyanate method, and the expression of angiogenic growth factors were detected by western blot analysis. We found that an 8-min or shorter period of ischemia induced neither permanent neurological deficits nor MRI detectable brain lesions in C57BL/6 mice, but histologically caused neuronal apoptosis and cerebral vasculature abnormalities. NBP treatment increased the number of CD31+ microvessels and perfused microvessels after TIA. NBP also up-regulated the expression of VEGF, Ang-1 and Ang-2 and improved the cerebrovascular network. In conclusion, 8 min or shorter cerebral ischemia induced by the suture MCAO method is an appropriate TIA model in C57BL/6 mice, which conforms to the definition of human TIA, but causes microscopic neurovascular impairment. NBP treatment increased the expression of angiogenic growth factors, promoted angiogenesis and improved cerebral microvessels after TIA. Our study provides new insights on the pathogenesis and potential treatments of TIA.

Genistein-3′-sodium sulfonate ameliorates cerebral ischemia injuries by blocking neuroinflammation through the α7nAChR-JAK2/STAT3 signaling pathway in rats

BackgroundNeuroinflammation plays a pivotal role in the acute progression of cerebral ischemia/reperfusion injury (I/RI). We previously reported that genistein-3′-sodium sulfonate (GSS), a derivative from the extract of the phytoestrogen genistein (Gen), protects cortical neurons against focal cerebral ischemia. However, the molecular mechanism underlying the neuroprotective effects exerted by GSS remains unclear. PurposeThe present study focused on the anti-inflammatory effects of GSS following I/RI in rats. Study designRandomized controlled trial. MethodsThe tMCAO rat model and LPS-stimulated BV2 in vitro model were used. Longa's scare was used to observe neurological function. TTC staining and Nissl staining were used to evaluate brain injury. ELISA, qRT-PCR, Western blotting and immunofluorescent staining methods were used to detect cytokine concentration, mRNA level, protein expression and location. ResultsGSS treatment improves neurological function, reduces the volume of cerebral infarction, attenuates proinflammatory cytokines and inactivates the phosphorylation of JAK2 and STAT3 in I/RI rats. Furthermore, GSS increased the expression of α7nAChR. More importantly, the neuroprotective, anti-inflammatory and inhibiting JAK2/STAT3 signaling pathway effects of GSS were counteracted in the presence of alpha-bungarotoxin (α-BTX), an α7nAChR inhibitor, suggesting that α7nAChR is a potential target associated with the anti-inflammatory effects of GSS in the I/RI rats. GSS also inhibited BV2 cells from releasing IL-1β via the α7nAChR pathway after LPS stimulation. ConclusionGSS protects against cerebral I/RI through the expression of α7nAChR and inhibition of the JAK2/STAT3 pathway. Our findings provide evidence for the role of the cholinergic anti-inflammatory pathway in neuroinflammation and uncover a potential novel mechanism for GSS treatment in ischemic stroke. The downstream signals of GSS, α7nAChR- JAK2/STAT3 could also be potential targets for the treatment of I/RI.

MicroRNAs sequencing of plasma exosomes derived from patients with atrial fibrillation: miR-124-3p promotes cardiac fibroblast activation and proliferation by regulating AXIN1.

MicroRNAs (miRNAs) play an important role in the pathogenesis of atrial fibrillation (AF). Exosomal miRNAs may develop as promising biomarkers for AF. To explore significant exosomal miRNAs in AF, plasma exosomes were extracted from 3 patients with AF and 3 patients with sinus rhythm (SR), respectively. Differential expression of exosomal miRNAs were screened by high-throughput sequencing analysis and verified by qRT-PCR from 40 patients with AF and 40 patients with SR. The target genes prediction, biological function, and signaling pathways analysis were conducted by miRanda software, gene ontology (GO), and KEGG analysis. The results showed that there were 40 differently expressed exosomal miRNAs from AF patients compared with SR patients, of which 13 miRNAs were upregulated and 27 miRNAs were downregulated. qRT-PCR validation demonstrated that miR-124-3p, miR-378d, miR-2110, and miR-3180-3p were remarkably upregulated, while miR-223-5p, miR-574-3p, miR-125a-3p, and miR-1299 were downregulated. To explore the function of miR-124-3p associated with AF, plasma exosomes derived from AF patients were co-incubated with rat myocardial fibroblasts. The expression of miR-124-3p was upregulated in myocardial fibroblasts. The viability and proliferation of myocardial fibroblasts were elevated by transfecting with miR-124-3p overexpression plasmids using CCK8 and immunofluorescence-staining methods. AXIN1 was verified to be the target of miR-124-3p by luciferase assay in vitro. Expression of AXIN1 was reduced, while β-catenin, Collagen 1, and α-SMA were increased in myocardial fibroblasts with miR-124-3p overexpression. In conclusion, these findings suggested that circulating exosomal miRNAs may serve as novel biomarkers for AF, and miR-124-3p promotes fibroblast activation and proliferation through regulating WNT/β-catenin signaling pathway via AXIN1.

Investigation of Ruminant Encephalitic and Septicemic Listeriosis by the Immunofluorescence Method

Listeria genus are Gram positive facultative intracellular bacteria that are found in the soil as saprophytes but also contain species that have pathogenic effects in their last host. Listeria monocytogenes is the major pathogen in Listeria species that is responsible for the majority of Listeriosis cases in humans and animals. In this study, it was aimed to investigate L. monocytogenes with histopathological and immunofluorescence methods in brain and liver tissues taken from sheep and cattle with clinical signs of suspicious Listeriosis. In the study, isolation and identification of L. monocytogenes with cultural methods were also applied. The material of the study consisted of 16 sheep and 2 bovine tissue samples. As a result of cultural analysis, L. monocytogenes was identified from 12 (66.6%) of the samples. Within the framework of histopathological and macroscopic findings, 16 (88.8%) of 18 cases with suspected Listeriosis were positive with the immunofluorescence method. These findings reveal that the immunofluorescent staining method is a reliable and sensitive method in the diagnosis of Listeriosis.

SIX1/EYA1 are novel liver damage biomarkers in chronic hepatitis B and other liver diseases.

BackgroundThis study aimed to investigate the clinicopathological significance of sine oculis homeobox homolog 1 (SIX1) and eyes absent 1 (EYA1) in patients with chronic hepatitis B (CHB) and other liver diseases.MethodsSIX1 and EYA1 levels were detected in human serum and liver tissues by enzyme linked immunosorbent assay (ELISA) and immunofluorescent staining method, respectively.ResultsThe serum SIX1 and EYA1 levels in 313 CHB patients were 7.24±0.11 and 25.21±0.51 ng/mL, respectively, and these values were significantly higher than those in 33 healthy controls (2.84±0.15 and 13.11±1.01 ng/mL, respectively; P<0.05). Serum SIX1 and EYA1 levels were also markedly increased in patients with numerous other liver diseases, including liver fibrosis, hepatocellular carcinoma, fatty liver disease, alcoholic liver disease, fulminant hepatic failure, autoimmune liver disease, and hepatitis C, compared to the healthy controls (P<0.05). Dynamic observation of these proteins over time in 35 selected CHB patients revealed that SIX1 and EYA1 serum levels increased over an interval. Immunofluorescent staining revealed that both SIX1 and EYA1 were only expressed in hepatic stellate cells (HSCs), and their increased expression was evident in CHB liver tissue.ConclusionsSIX1 and EYA1 are novel biomarkers of liver damage in patients of CHB and other liver diseases, with potential clinical utility.

Elucidation of Regional Mechanical Properties of Brain Tissues Based on Cell Density

Research on the mechanical properties of brain tissue has received extensive attention. However, most of the current studies have been conducted at the phenomenological level. In this study, the indentation method was used to explore the difference in local mechanical properties among different regions of the porcine cerebral cortex. Further, hematoxylin-eosin and immunofluorescence staining methods were used to determine the correlation between the cellular density at different test points and mechanical properties of the porcine cerebral cortex. The frontal lobe exhibited the strongest viscosity. The temporal lobe displayed the lowest sensitivity to changes in the indentation speed, and the occipital lobe exhibited the highest shear modulus. Additionally, the shear modulus of different areas of the cerebral cortex was negatively correlated with the total number of local cells per unit area and positively correlated with the number of neuronal cell bodies per unit area. Exploration of the mechanical properties of the local brain tissue can provide basic data for the establishment of a finite element model of the brain and mechanical referential information for the implantation position of brain chips.

The Protective Effects of Sulforaphane on High-Fat Diet-Induced Obesity in Mice Through Browning of White Fat.

Background: Sulforaphane (SFN), an isothiocyanate naturally occurring in cruciferous vegetables, is a potent indirect antioxidant and a promising agent for the control of metabolic disorder disease. The glucose intolerance and adipogenesis induced by diet in rats was inhibited by SFN. Strategies aimed at induction of brown adipose tissue (BAT) could be a potentially useful way to against obesity. However, in vivo protective effect of SFN against obesity by browning white adipocyte has not been reported. Our present study is aimed at evaluation the efficacy of the SFN against the high-fat induced-obesity mice and investigating the potential mechanism. Methods: High-Fat Diet-induced obese female C57BL/6 mice were intraperitoneally injected with SFN (10 mg/kg) daily. Body weight was recorded every 3 days. 30 days later, glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed. At the end of experiment, fat mass were measured and the adipogenesis as well as browning associated genes expression in white adipose tissue (WAT) were determined by RT-qPCR and western blot. Histological examination of the adipose tissue samples were carried out with hematoxylin–eosin (HE) staining and immunofluorescence staining method. In vitro, pre-adipocytes C3H10T1/2 were treated with SFN to investigate the direct effects on adipogenesis. Results: SFN suppressed HFD-induced body weight gain and reduced the size of fat cells in mice. SFN suppressed the expression of key genes in adipogenesis, inhibited lipid accumulation in C3H10T1/2 cells, increased the expression of brown adipocyte-specific markers and mitochondrial biogenesis in vivo and in vitro, and decreased cellular and mitochondrial oxidative stress. These results suggested that SFN, as a nutritional factor, has great potential role in the battle against obesity by inducing the browning of white fat. Conclusion: SFN could significantly decrease the fat mass, and improve glucose metabolism and increase insulin sensitivity of HFD-induced obese mice by promoting the browning of white fat and enhancing the mitochondrial biogenesis in WAT. Our study proves that SFN could serve as a potential medicine in anti-obesity and related diseases.

Detecting Amyloid-&amp;#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-β. In this protocol, we describe the method of immunofluorescent staining in 5×FAD transgenic mouse to detect amyloid-β accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Detecting Amyloid-β Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-β (Aβ) in the brain. Aβ is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. In pathological circumstances, the increased β-cleavage of APP leads to overproduction of Aβ, which aggregates into Aβ plaques. Since Aβ plaques are a characteristic of AD pathology, detecting the amount of Aβ is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize Aβ deposition. The mouse model used in our experiments is 5×FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study Aβ pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect Aβ accumulation in 5×FAD mice. With this protocol, researchers can investigate Aβ pathology in an AD mouse model.

Neuroprotection of Heme Oxygenase-2 in Mice AfterIntracerebral Hemorrhage.

There are few effective preventive or therapeutic strategies to mitigate the effects of catastrophic intracerebral hemorrhage (ICH) in humans. Heme oxygenase is the rate-limiting enzyme in heme metabolism; heme oxygenase-2 (HO-2) is a constitutively expressed heme oxygenase. We explored the involvement of HO-2 in a collagenase-induced mouse model of ICH in C57BL/6 wild-type and HO-2 knockout mice. We assessed oxidative stress injury, blood-brain barrier permeability, neuronal damage, late-stage angiogenesis, and hematoma clearance using immunofluorescence, Western blot, MRI, and special staining methods. Our results show that HO-2 reduces brain injury volume and brain edema, alleviates cytotoxic injury, affects vascular function in the early stage of ICH, and improves hematoma absorbance and angiogenesis in the late stage of ICH in this model. Thus, we found that HO-2 has a protective effect on brain injury after ICH.

Sensitivity of a novel immunofluorescent staining method for Circulating tumor cells identification and its clinical application in predicting cancer recurrence in breast cancer

Circulating tumor cells (CTC) enumeration has been clinically adopted as important prognostic factor in different cancers, but many of CTCs isolation or identification apporches did not have clinical data. In this study, we developed a novel indirect immunofluorescent staining method targeting EpCAM or cytokeratin for CTC identification. Peripheral blood samples from twenty healthy donors and thirty-six cancer patients were used for cutoff establishment and sensitivity test. An immunofluorescent intensity threshold value of 5080 for the EpCAM or cytokeratin staining led to highly accurate cell classification (95% sensitivity and &gt;99% specificity), and with the cut-off point of 2 CTCs, the sensitivity of CTC detection was 54.2% (19/35) in the diagnosis of patients with different cancer. Besides, the presence of CTCs defined as having &gt;2 CTCs/7.5 mL might be associated with cancer staging and occurred three weeks earlier than cancer relapse in a breast cancer patient, and thus was consider a clinically useful tool to monitor tumor recurrence during chemotherapy.