Anti-mouse Secondary Antibody Research Articles

Overexpression of ASPH protein predicts poor outcomes in retroperitoneal liposarcoma patients.

Overexpression of ASPH protein predicts poor outcomes in retroperitoneal liposarcoma patients.

Inducing ciliation in equine oviduct epithelial cell monolayers

Recently, we developed re-differentiated equine oviduct epithelial cell (EOEC) monolayers in Transwell inserts. EOEC monolayers demonstrated various in vivo-like morphological characteristics, but often lacked secondary cilia. The monolayers were classified as low (<1% secondary cilia) or high (>5% secondary cilia) spontaneously re-differentiated, with the latter in the minority (5-10%). Interestingly, only high re-differentiated monolayers responded to Notch inhibition by developing in vivo-like secondary ciliation rates, i.e. over 50% ciliated cells. In this study, we aimed to consistently generate high re-differentiated monolayers. We first assessed whether secondary cilia formation was affected by donor mare, cycle stage and/or experiment. For this, harvested oviduct explants were cultured for 10 days at a concentration of 10 × 106 EOECs per 9.6 cm² well, to yield a confluent de-differentiated monolayer in conventional wells. Subsequently, the monolayers were trypsinized and the EOECs were reseeded onto the microporous membrane of hanging inserts at a concentration of 0.3 × 105 EOECs per 0.33 cm² insert. Three days after seeding, the EOECs were triggered to re-differentiate by air-liquid interface introduction (ALI). After 1 and 2 months in culture, EOEC monolayers were fixed and their phenotype was assessed by combined immunofluorescence staining to detect cilia (mouse anti-acetylated α-tubulin antibody; Alexa 488-conjugated goat anti-mouse secondary antibody),cell nuclei (Hoechst 33342) and actin filaments (Alexa 568-conjugated phalloidin). Neither cell donor nor cycle stage had any effect on secondary ciliation rates, whereas experimental modifications did. In 3/15 experiments, >95% of the monolayers demonstrated high re-differentiated monolayers. To discover the source of experimental variation, the seeding concentration of the de-differentiated EOECs was varied between 0.1 and 10 × 105 EOECs per 0.33 cm² insert; this did not affect secondary ciliation rates. In a third experiment, the effect of varying the seeding concentration of the harvested oviduct explant EOECs used to establish the de-differentiated monolayers, between 1 and 30 × 106 EOECs per 9.6 cm² well, was evaluated. EOECs harvested from de-differentiated monolayers 10 days after oviduct explant seeding at 1 or 5 × 106 EOECs per well supported the formation of high re-differentiated monolayers in >95% of cases. Within 1 month after ALI, >50% and >30% of the re-differentiated EOECs showed secondary cilia after initial seeding of 1 and 5 × 106 oviduct explant EOECs per well, respectively. Notch-inhibition helped to further increase secondary ciliation rates to >70% in the re-differentiated monolayers. We conclude that initial oviduct explant EOEC seeding concentration is critical to subsequently generating monolayers that closely resemble in vivo oviduct epithelia.

Tissue Tropism of H9N2 Low-Pathogenic Avian Influenza Virus in Broiler Chickens by Immunohistochemistry

The H9N2 subtype of low-pathogenic avian influenza viruses (LPAIV) is a widespread pathogen of poultry that can also infect humans. The characterization of viral infections is a complex process, involving clinical, pathological, and virological investigations. The aim of this study was to adapt and optimize an immunohistochemical (IHC) technique developed for LPAIVs specifically for the detection of H9N2 virus antigens in infected tissues. Twenty-one-day-old broiler chickens were inoculated with three different strains of H9N2 virus by different infection routes (i.e., intranasal-intratracheal and intravenous) or co-infected with infectious bronchitis virus (IBV) and observed for 11 days post infection. The suggested IHC protocol was modified: (i) DAB (diamino-benzidine) was substituted with AEC (3-amino-9-ethyl carbazole) as chromogen; and (ii) indirect two-step immune reactions of monoclonal primary and peroxidase-labeled anti-mouse secondary antibodies were used instead of avidin-biotin complexes. Avian influenza virus antigen appears as a red precipitate in the nuclei of affected cells but can also be identified in the cytoplasm. Mild hyperemia and congestion were observed in the trachea, air sac, and lungs of the challenged birds, and fibrinous exudate was found at the bifurcation in a few cases. Neither gross pathological nor IHC lesions were found in the control group. Using the optimized protocol and an associated scoring scheme, it was demonstrated that the H9N2 strains tested exhibited respiratory and urinary tract tropism irrespective of the route of inoculation. On day 5, viral antigen was detected in the respiratory tract and kidney in 30-50% of the samples. On day 11, no IHC signal was observed, indicating the lack of viral replication. Slight differences in viral antigen expression were found between the different H9N2 virus strains, but, in contrast to highly pathogenic avian influenza (HPAI), no viral antigen was detected in the brain and pancreas. Thus, IHC can be considered as an informative, visual addition to the toolkit for the characterization of H9N2 LPAIV infections.

Tellurium-containing polymer for mass cytometry

Mass cytometry is a powerful analytical technique for high-dimensional single-cell analysis. In this technique, cells are stained with heavy isotope-tagged antibodies and are analyzed by an inductively coupled plasma time-of-flight mass spectrometer. While the current mass cytometer has more than 100 mass channels, only about 50 are used because reagents are only available for a fraction of the useful elements. Here we describe the synthesis of a tellurium-containing polymer based on poly-l-lysine and sulfated tellurophene. The polymer carried ca. 48 tellurophene units per chain and had a terminal azide group for antibody conjugation. While the polymer could be conjugated to antibodies, the resulting conjugates gave mixed results in mass cytometry immunoassays. For example, a goat anti-mouse (secondary) antibody and an anti-CD19 primary Ab labeled with polymers gave promising results in antibody titration experiments. However, an anti-CD20 Ab labeled with a 126Te-enriched polymer no longer recognized the antigen and showed significant non-specific binding in a 7-plex assay with human PBMCs.

Development of a colloidal gold immunochromatographic strip for the rapid detection of antibodies against Fasciola gigantica in buffalo

BackgroundFasciola gigantica, a tropical liver fluke, infects buffalo in Asian and African countries, causing significant economic losses and posing public health threats. The diagnostic of buffalo fascioliasis caused by F. gigantica is vital in fascioliasis control and preventation. The 22nd gel filtration chromatography fraction of F. gigantica Excretory-Secretory Products (FgESP), namely Fasciola 22 (F22), which was used as a diagnostic antigen in indirect ELISA, has demonstrated great potential for fascioliasis diagnosing. In the absence of rapid diagnostic methods, the use of a colloidal gold immunochromatographic strip based on F22 was applied to detect F. gigantica infection in buffalo.MethodsIn the present study, the 22nd gel filtration chromatography fraction of FgESP (F22) was used as an antigen to establish the colloidal gold-based immunochromatographic strip (ICS). The nitrocellulose membrane was incubated with F22 at the test line (T line) and goat anti-mouse secondary antibody at the control line (C line). The mouse anti-buffalo secondary antibody 2G7 conjugated to colloidal gold particles was used as the detection system for line visualization. The strip was assembled and developed by optimizing reaction conditions. The sensitivity, specificity, stability, and early diagnostic value of the strip were evaluated employing buffalo-derived sera.ResultsAn immunochromatographic strip for the rapid detection of antibodies against F. gigantica-FgICS was developed. The strip demonstrated high sensitivity and specificity. Sensitivity tests confirmed positive results even when the positive reference serum was diluted 4,096 times. Except for one Schistosoma japonicum-positive serum that tested positive via FgICS, specificity tests confirmed no cross-reactivity with other positive sera of Schistosoma japonicum and Babesia bovis. The strip remained stable after storage at 4°C for up to 3 months. In infected buffalo, antibodies could be detected as early as 14–21 days post-infection. The detection of 17 positive sera yielded an 82.4% positive rate via FgICS vs. a 100.0% positive rate via ELISA based on FgESP. For FgICS, the 95% confidence interval of sensitivity was 84.8–95.4%, while specificity was 4.2–14.7%.ConclusionThe immunochromatographic strip FgICS developed in this study provides a simple and rapid method of F. gigantica antibody detection and infected buffalo monitoring in the field.

A Novel Prototype Biosensor Array Electrode System for Detecting the Bacterial Pathogen Salmonella typhimurium.

Salmonellosis caused by Salmonella sp. has long been reported all over the world. Despite the availability of various diagnostic methods, easy and effective detection systems are still required. This report describes a dialysis membrane electrode interface disc with immobilized specific antibodies to capture antigenic Salmonella cells. The interaction of a specific Salmonella antigen with a mouse anti-Salmonella monoclonal antibody complexed to rabbit anti-mouse secondary antibody conjugated with HRP and the substrate o-aminophenol resulted in a response signal output current measured using two electrode systems (cadmium reference electrode and glassy carbon working electrode) and an agilent HP34401A 6.5 digital multimeter without a potentiostat or applied potential input. A maximum response signal output current was recorded for various concentrations of Salmonella viz., 3, 30, 300, 3000, 30,000 and 300,000 cells. The biosensor has a detection limit of three cells, which is very sensitive when compared with other detection sensors. Little non-specific response was observed using Streptococcus, Vibrio, and Pseudomonas sp. The maximum response signal output current for a dialysis membrane electrode interface disc was greater than that for gelatin, collagen, and agarose. The device and technique have a range of biological applications. This novel detection system has great potential for future development and application in surveillance for microbial pathogens.

In vitro evolution of myc-tag antibodies: in-depth specificity and affinity analysis of Myc1-9E10 and Hyper-Myc.

One of the most widely used epitope tags is the myc-tag, recognized by the anti-c-Myc hybridoma antibody Myc1-9E10. Combining error-prone PCR, DNA shuffling and phage display, we generated an anti-c-Myc antibody variant (Hyper-Myc) with monovalent affinity improved to 18nM and thermal stability increased by 37%. Quantification of capillary immunoblots and by flow cytometry demonstrated improved antigen detection by Hyper-Myc. Further, three different species variants of this antibody were generated to allow the use of either anti-human, anti-mouse or anti-rabbit Fc secondary antibodies for detection. We characterized the specificity of both antibodies in depth: individual amino acid exchange mapping demonstrated that the recognized epitope was not changed by the in vitro evolution process. A laser printed array of 29,127 different epitopes representing all human linear B-cell epitopes of the Immune Epitope Database allowing to chart unwanted reactivities with mimotopes showed these to be very low for both antibodies and not increased for Hyper-Myc despite its improved affinity. The very low background reactivity of Hyper-Myc was confirmed by staining of myc-tag transgenic zebrafish whole mounts. Hyper-Myc retains the very high specificity of Myc1-9E10 while allowing myc-tag detection at lower concentrations and with either anti-mouse, anti-rabbit or anti human secondary antibodies.

Development of Human Gut Organoids With Resident Tissue Macrophages as a Model of Intestinal Immune Responses.

Development of Human Gut Organoids With Resident Tissue Macrophages as a Model of Intestinal Immune Responses.

Abstract MP11: Decreased Expression Of Ace2 Is Associated Urine Derived Human Renal Proximal Tubule Cells From Inverse Salt Sensitivity Participants Cultured In Low Salt Conditions

Increased morbidity and mortality occurs in some individuals consuming low sodium diets. Inverse salt sensitivity (ISS) is the paradoxical increase in blood pressure of individuals to a low sodium diet. Our group previously reported decreased expression of dopamine type 2 receptor (D 2 R), increased expression Aminopeptidase N, and increased Ang II dependent sodium transport in human urine derived renal proximal tubule cells isolated from ISS participants. In an attempt to understand the increased Ang II sensitivity demonstrated in ISS cells, we examined angiotensin converting enzyme 2 (ACE2), a membrane associated enzyme involved in the metabolism of Ang II. Urine derived renal proximal tubule cells grown and immortalized from ISS participants were compared to cells from salt resistant (SR) participants cultured in iso-osmotic media with low salt (LS, 90 mM NaCl) normal salt (NS, 140 mM NaCl) and high salt (HS, 190 mM NaCl). Cells were incubated in LS, NS, and HS media with and without losartan (LOS,1 μM) overnight (18 hours) and ACE2 expression levels determined by in-cell western blot. A monoclonal antibody specific to an extracellular epitope of ACE2 was used as the primary antibody and an Alexa-647 anti-mouse secondary antibody. ACE2 expression was only reduced in ISS cells in LS condition (28.7±2.1 % reduction, ISS LS vs SR LS, N=4 per group, p&lt;0.05). Addition of losartan completely blocked the decrease in ACE2 expression in low salt conditions in ISS in urine derived human renal proximal tubule cells. No other changes in ACE2 expression were found between ISS and SR in either NS or HS conditions and with or without losartan. In conclusion, a decreased expression of ACE2 in ISS urine proximal tubule cells could explain the previously reported increased sensitivity of ISS cells to Ang II by increasing the half-life of Ang II under low salt conditions.

An Automated Multicycle Immunoaffinity Enrichment Approach Developed for Sensitive Mouse IgG1 Antibody Drug Analysis in Mouse Plasma Using LC/MS/MS.

In the immuno-oncology field, surrogate mouse monoclonal antibodies are often preferred in establishing proper PK/PD/efficacy correlations as well as supporting anticipated mouse to human translation. Thus, a highly sensitive and specific bioanalytical method is needed in quantifying those surrogate mouse antibodies after dosing in mice. Unfortunately, when specific reagents, such as recombinant target antigen and anti-idiotypic antibody, are not available, measuring mouse surrogate antibody drugs in mice is very challenging for ligand binding assay (LBA) due to the severe cross reactivity potential. Different from LBA, if at least one unique surrogate peptide can be identified from the surrogate antibody sequence, the immunoaffinity enrichment based LC/MS/MS assay may be able to differentiate the analyte response from the high endogenous immunoglobulin background and provide adequate sensitivity. Herein, a new automated multicycle immunoaffinity enrichment method was recently developed to extract a surrogate mouse IgG1 (mIgG1) antibody drug from mouse plasma using a commercially available antimouse IgG1 secondary antibody. In the assay, reuse of the capture antibody up to six times mostly resolved the binding capacity issue caused by the abundant endogenous mIgG1 and made the immunoaffinity enrichment step more cost-effective. Combined with a unique surrogate peptide identified from the antibody, the LC/MS/MS assay achieved a limit of quantitation of 5 ng/mL with satisfactory assay precision, accuracy, and dynamic range. The successful implementation of this novel approach in discovery pharmacokinetic (PK) studies eliminates the dependence on specially generated immunoaffinity capturing reagents.

A Rapid Immunochromatographic Method Based on a Secondary Antibody-Labelled Magnetic Nanoprobe for the Detection of Hepatitis B preS2 Surface Antigen.

Hepatitis B is a globally prevalent viral infectious disease caused by the hepatitis B virus (HBV). In this study, an immunochromatographic assay (ICA) for the rapid detection of hepatitis B preS2 antigen (preS2Ag) was established. The magnetic nanoparticles (MNPs) indirectly labelled with goat anti-mouse (GAM) secondary antibody were applied as a nanoprobe for free preS2 antibody (preS2Ab) capturing and signal amplification. By employing sample pre-incubation processing as well, preS2Ag-preS2Ab was sufficiently caught by the GAM-MNPs probe in 5 min. A qualitative sensitivity of 625 ng/mL was obtained by naked-eye observation within 15–20 min. A standard curve (0–5000 ng/mL) was established, with a quantitative limit of detection (LOD) of 3.6 ng/mL, based on the stability and penetrability of the magnetic signal characteristics. The proposed method for preS2Ag was rapid (~25 min, cf. ELISA ~4 h) and had a good accuracy, which was verified using an ELISA kit (relative error < 15%). Large equipment and skilled technicians were not required. The sensitivity and specificity of the developed GAM-MNPs-ICA method were 93.3% and 90% in clinical serum samples (n = 25), respectively. A good detection consistency (84%) was observed between the developed ICA method and 2 types of commercial ELISA kits, indicating that the GAM-MNPs-ICA has a potential application in large-scale screening for and point-of-care diagnosis of hepatitis B or other infectious diseases.

Tackling the Mouse-on-Mouse Problem in Cochlear Immunofluorescence: A Simple Double-Blocking Protocol for Immunofluorescent Labeling of Murine Cochlear Sections with Primary Mouse Antibodies.

The mouse is the most widely used animal model in hearing research. Immunohistochemistry and immunofluorescent staining of murine cochlear sections have, thus, remained a backbone of inner ear research. Since many primary antibodies are raised in mouse, the problem of "mouse-on-mouse" background arises due to the interaction between the anti-mouse secondary antibody and the native mouse immunoglobulins. Here, we describe the pattern of mouse-on-mouse background fluorescence in sections of the postnatal mouse cochlea. Furthermore, we describe a simple double-blocking immunofluorescence protocol to label mouse cochlear cryosections. The protocol contains a conventional blocking step with serum, and an additional blocking step with a commercially available anti-mouse IgG blocking reagent. This blocking technique virtually eliminates the "mouse-on-mouse" background in murine cochlear sections, while adding only a little time to the staining protocol. We provide detailed instructions and practical tips for tissue harvesting, processing, and immunofluorescence-labeling. Further protocol modifications are described, to shorten the duration of the protocol, based on the primary antibody incubation temperature. Finally, we demonstrate examples of immunofluorescence staining performed using different incubation times and various incubation temperatures with a commercially available mouse monoclonal primary antibody. © 2020 The Authors. Basic Protocol: Tackling the Mouse-on-Mouse Problem in Cochlear Immunofluorescence: A Simple Double-Blocking Protocol for Immunofluorescent Labeling of Murine Cochlear Sections with Primary Mouse Antibodies.

Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos.

5-methylcytosine (5mC) is an epigenetic modification to DNA which modulates transcription. 5mC can be sequentially oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Collectively, these marks are referred to as the oxidized derivatives of 5mC (i.e., oxi-mCs). Their formation is catalyzed by the ten-eleven translocation methylcytosine dioxygenases (TETs 1, 2 and 3). Various techniques have been developed for the detection of oxi-mCs. The following chapter describes an immunochemical protocol for the simultaneous detection of 5hmC and 5caC in embryonic zebrafish tissue sections. The embryos are fixed, permeabilized and embedded in paraffin blocks. The blocks are cut into sections that are mounted onto slides. Depurination of the DNA is performed to allow immunodetection of the oxi-mCs. The 5hmC is detected with the help of a mouse anti-5hmC monoclonal primary antibody and a goat anti-mouse Alexa Fluor 633-conjugated secondary antibody. The weak 5caC signal requires enzymatic amplification. Its detection involves a rabbit anti-5caC polyclonal primary antibody and a goat anti-rabbit secondary antibody that is conjugated to horseradish peroxidase (HRP). HRP amplifies the 5caC signal by catalyzing the deposition of large quantities of fluorescein-labeled tyramide. Sections immunostained for 5hmC and 5caC are analyzed by fluorescent light or confocal laser scanning microscopy. This immunochemical method allows for highly sensitive detection of 5hmC and 5caC in zebrafish tissues.

The Accumulation of Tau-Immunoreactive Hippocampal Granules and Corpora Amylacea Implicates Reactive Glia in Tau Pathogenesis during Aging.

SummaryThe microtubule-associated tau protein forms pathological inclusions that accumulate in an age-dependent manner in tauopathies including Alzheimer's disease (AD). Since age is the major risk factor for AD, we examined endogenous tau species that evolve during aging in physiological and diseased conditions. In aged mouse brain, we found tau-immunoreactive clusters embedded within structures that are reminiscent of periodic acid-Schiff (PAS) granules. We showed that PAS granules harbor distinct tau species that are more prominent in 3xTg-AD mice. Epitope profiling revealed hypo-phosphorylated rather than hyper-phosphorylated tau commonly observed in tauopathies. High-resolution imaging and 3D reconstruction suggest a link between tau clusters, reactive astrocytes, and microglia, indicating that early tau accumulation may promote neuroinflammation during aging. Using postmortem human brain, we identified tau as a component of corpora amylacea (CA), age-related structures that are functionally analogous to PAS granules. Overall, our study supports neuroimmune dysfunction as a precipitating event in tau pathogenesis.

Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method.

.Three-dimensional visualization of the innervation in skeletal muscles is helpful for understanding the morphological structure and function. iDISCO, a whole-mount immunolabeling and clearing technique, provides a valuable tool for volume imaging of intramuscular nerve fibers but suffers from the nonspecific staining caused by the anti-mouse secondary antibody when using the murine primary antibody. We developed a modified iDISCO method by introducing pretreatment of ScaleCUBIC-1 reagent, termed m-iDISCO. The m-iDISCO method could eliminate the nonspecific staining and achieve uniform and complete labeling of nerve fibers in various muscles with mouse anti-neurofilament primary antibody. Combining the m-iDISCO method with light-sheet microscopy enabled us to visualize the innervation of adult mouse tibialis anterior and trace the nerve fibers from extramuscular branches to intramuscular terminal branches. This method represents an effective alternative for studying the innervation of intact skeletal muscles in health and disease.

CAR T‐cells' Interaction with Artificial Antigen Presenting Cell Surface

Adoptive Cell Transfer (ACT) using Chimeric Antigen Receptor (CAR) T-cells shows promising results in treating metastatic melanoma and B cell malignancies. However, the detailed mechanism of CAR T-cell's interaction with tumors is still not well-understood. In this work, we created an artificial antigen presenting cell (APC) surface for 2nd generation (CD3 and CD28 signaling domains) CD19 and GD2 CAR T-cells and studied the interaction of CAR T-cells in terms of synapse formation. We created circular patterns of antibodies against CAR T-cells using micro-contact printing and measured the level of activation and degranulation in both CAR T-cells by quantifying the intensity of the phosphorylated CD3ζ chain (p-CD3ζ) and the Lysosome-Associated Membrane Protein 1 (LAMP-1) respectively. The results show that we can study the interaction of CAR T cell with APC surface in a reproducible manner and that different CAR T cells have a different level of activation and degranulation. Our findings suggest that the artificial APC allows for quantitative measurement of effectiveness for different CAR T cells with controlled APC density. Support or Funding Information This work was supported by the National Science Foundation through the Early-concept Grants for Exploratory Research (EAGER) (Award number: 1649243) and was performed in part at the Joint School of Nanoscience and Nanoengineering, a member of the Southeastern Nanotechnology Infrastructure Corridor (SENIC) and National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (Grant ECCS-1542174). The figure shows the interaction of GD2 CAR T-cell with 5 μm micropatterns of 1A7 (target). The aggregation of the phosphorylated CD3 zeta chain (p-CD3ζ) at the contact with the micropattern is clear. Micropatterns of 1A7 are labeled with Alexa Fluor 568 Goat anti-mouse secondary antibody and p-CD3ζ is labeled with Alexa Fluor 488 primary antibody. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Construction of Electrochemical Immunosensor Based on Gold-Nanoparticles/Carbon Nanotubes/Chitosan for Sensitive Determination of T-2 Toxin in Feed and Swine Meat.

T-2 toxin (T-2) is one of major concern mycotoxins acknowledged as an unavoidable contaminant in human foods, animal feeds and also agriculture products. Thus, a facile and sensitive method is essential for accurate T-2 toxin detection. In our work, a specific electrochemical immunosensor based on gold nanoparticles/carboxylic group-functionalized single-walled carbon nanotubes/chitosan (AuNPs/cSWNTs/CS) composite was established. The mechanism of the electrochemical immunosensor was an indirect competitive binding to a given amount of anti-T-2 between free T-2 and T-2-bovine serum albumin, which was conjugated on covalently functionalized cSWNTs decorated on the glass carbon electrode. Afterwards, the alkaline phosphatase labeled anti-mouse secondary antibody was bound to the electrode surface by reacting with the primary antibody. Lastly, alkaline phosphatase catalyzed the hydrolysis of the substrate α-naphthyl phosphate, which produced an electrochemical signal. Compared with conventional methods, the established immunosensor was more sensitive and simpler. Under optimal conditions, this method could quantitatively detect T-2 from 0.01 to 100 μg·L−1 with a detection limit of 0.13 μg·L−1 and favorable recovery 91.42–102.49%. Moreover, the immunosensor was successfully applied to assay T-2 in feed and swine meat, which showed good correlation with the results obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS).

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Abstract 3831: The development of an immunohistochemical digoxigenin-labeled mouse on mouse single and double stain methodology

Abstract Introduction When a mouse or monoclonal antibody is desired for immunohistochemical detection on mouse tissues, the anti-mouse secondary antibody will also bind to endogenous mouse IgG in the tissue and may produce unwanted background staining. In the past, mouse-on-mouse (MM) detection utilized a biotinylated primary antibody or biotinylation kit. Tissues rich in endogenous biotin such as kidney or liver made it difficult to use, as streptavidin-horseradish peroxidase is required for detection. Attempts to completely negate endogenous biotin are ineffective as avidin-biotin blocking steps are unlikely to work acceptably in problematic tissues. Therefore, the design of a digoxigenin-labeled detection system is advantageous for MM detection as it would eliminate these issues. This technology has broad application as it allows the use of a variety of monoclonal antibodies reactive on mouse tissues. Materials and Methods Mouse tissues were formalin-fixed for 24 hours and embedded in paraffin (FFPE). Sections were cut and dried on slides before deparaffinizing and peroxidase blocking in the usual manner. Slides were then HIER in a citrate-based buffer prior to application of antibody. Mouse antibody concentrates were selected and mixed with a Digoxigenin Anti-Mouse Linker and incubated at room temperature (RT) for 1 hour. An Absorption Reagent is then added to the mixture, and further incubated for 30 minutes. The mixture is then diluted in antibody diluent to the desired concentration and applied to the mouse tissue for 60 minutes at RT, followed by a rabbit anti-digoxigenin antibody for 15 minutes. Detection of anti-digoxigenin is performed with an incubation in either a goat anti-rabbit HRP or AP Polymer for 30 minutes. Visualization is accomplished by using a DAB or Fast Red chromogen. For double stains, a sequential DS method using two monoclonal antibodies was employed using a heating step in retrieval solution after the first chromogenic reaction followed by a second monoclonal antibody and detection with either a rabbit-AP or HRP polymer. Visualization of the second antibody target is accomplished by using Fast Red or a blue chromogen. Slides were coverslipped with a permanent mounting medium and evaluated using brightfield microscopy. Results MM single stains with a variety of antibodies were easily achieved with specific, clean and biotin-free staining. Sequential DS techniques using monoclonal antibodies were also achieved and visualized with DAB, Fast Red or blue chromogens. Both manual and automated procedures were used. Conclusion The replacement of biotin-labeled MM technology with a digoxigenin MM technology is a superior method that not only eliminates endogenous biotin, but allows utilization of HRP and AP polymers for double stain technology. Citation Format: Joseph Vargas, David Tacha, Sara Figueroa. The development of an immunohistochemical digoxigenin-labeled mouse on mouse single and double stain methodology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3831. doi:10.1158/1538-7445.AM2017-3831

High-Throughput Screening of Sulfated Proteins by Using a Genome-Wide Proteome Microarray and Protein Tyrosine Sulfation System.

Protein tyrosine sulfation (PTS) is a widespread posttranslational modification that induces intercellular and extracellular responses by regulating protein-protein interactions and enzymatic activity. Although PTS affects numerous physiological and pathological processes, only a small fraction of the total predicted sulfated proteins has been identified to date. Here, we localized the potential sulfation sites of Escherichia coli proteins on a proteome microarray by using a 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis. Among the 4256 E. coli K12 proteins, 875 sulfated proteins were identified using antisulfotyrosine primary and Cy3-labeled antimouse secondary antibodies. Our findings add considerably to the list of potential proteins subjected to tyrosine sulfation. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome microarrays, and we expect such approaches to contribute substantially to the understanding of important human diseases.