Journal: JCI Insight

Article Title: Anti-GRP78 autoantibodies induce endothelial cell activation and accelerate the development of atherosclerotic lesions

doi: 10.1172/jci.insight.99363

Figure Lengend Snippet: ER stress causes an increase in csGRP78 levels on ECs. HAECs were treated with ( A ) 2.5 μg/ml Tm or ( B ) 300 nM Tg for the indicated time period or with ( C ) 7-ketocholesterol (7KC), Sin-1, 4-hydroxynonenal (4HNE), or peroxynitrite (Peroxy) for 8 hours, after which surface proteins were biotinylated and separated by streptavidin pull down. Total cell lysates (tGRP78) and surface protein fractions (sGRP78) were subjected to Western blotting for detection of GRP78, the EC marker CD31, IRE1α, or phospho-eIF1α. Blots were also immunostained for β actin as a protein loading control. Densitometry quantification is indicated under each band relative to untreated cells and normalized to β-actin.

Article Snippet: Following antigen retrieval where necessary, sections were blocked with 5% normal serum, and incubated with the primary antibody for 1 hour, followed by biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) diluted 1:500, and streptavidin HRP (Vector Laboratories) diluted 1:50.

Techniques: Western Blot, Marker

Journal: Analytical chemistry

Article Title: Surface Plasmon Resonance (SPR) Detection Using Antibody-Linked Magnetic Nanoparticles for Analyte Capture, Purification, Concentration and Signal Amplification

doi: 10.1021/ac900007c

Figure Lengend Snippet: (A) Stepwise detection in PBS-T buffer of (1) 10 ng/mL SEB followed by signal amplification/verification with (2) 50 μg/mL anti-SEB (monoclonal, biotinylated) and further amplification by (3) streptavidin/colloidal IMB nanoparticle conjugates

Article Snippet: The unbiotinylated monoclonal anti-SEB antibodies served as a control and exhibited no avidin binding, while the commercially biotinylated antibodies from Vector Labs and the biotinylated monoclonal anti-SEB each bound similar amounts of avidin.

Techniques: Amplification

Journal: European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology

Article Title: Effects of cocaine history on postsynaptic GABA receptors on dorsal raphe serotonin neurons in a stress-induced relapse model in rats

doi: 10.1016/j.euroneuro.2015.11.009

Figure Lengend Snippet: GABA synaptic activity is selectively sensitized in DRN-5-HT neurons following yohimbine stress-induced reinstatement of cocaine-seeking. (a) Immunohistochemical identification of the biocytin-filled recorded neuron (green) with TPH2-IR (red) is shown

Article Snippet: Biocytin was visualized using Alexa 488-conjugated streptavidin (1:500; Life Technologies, Carlsbad, CA).

Techniques: Activity Assay, Immunohistochemistry

Journal: European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology

Article Title: Effects of cocaine history on postsynaptic GABA receptors on dorsal raphe serotonin neurons in a stress-induced relapse model in rats

doi: 10.1016/j.euroneuro.2015.11.009

Figure Lengend Snippet: GABA synaptic activity is unchanged in DRN- non-5-HT neurons following yohimbine stress-induced reinstatement of cocaine-seeking. (a) Immunohistochemical identification of the biocytin-filled recorded neuron (green) in a non-5-HT neuron without TPH-IR

Article Snippet: Biocytin was visualized using Alexa 488-conjugated streptavidin (1:500; Life Technologies, Carlsbad, CA).

Techniques: Activity Assay, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)—Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV

doi: 10.3390/ijms21072353

Figure Lengend Snippet: Distribution of avian influenza virus NP in selected organs of inoculated birds. Distribution of influenza NP in brain ( A , E , I , M ), lung ( B , F , J , N ), spleen ( C , G , K , O ) and heart ( D , H , L , P ) of inoculated chickens of selected viruses at 4 dpi (except for H5N1_H4_T 327 K) as detected by immunohistochemistry using primary polyclonal rabbit anti-NP A/FPV/Rostock/34 antibody (1:750) and a secondary biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) antibody (1:200). 3-amino-9-ethyl-carbazol (red-brown); hematoxylin counterstain (blue); Nomarski contrast; bars A,B,D,E,F,H,I,J,L,M,N,P = 20 µm. Bars C,G,K,O = 50 µm.

Article Snippet: Following sections were used for immunohistochemistry using the avidin–biotin–peroxidase complex method (Vector Laboratories Burlingame, CA, USA) with a primary polyclonal rabbit anti-NP antibody (1:750), and a secondary biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) antibody (1:200) as described [ , ].

Techniques: Immunohistochemistry, Plasmid Preparation

Journal: British Journal of Cancer

Article Title: Antibodies to CD20 and MHC class II antigen bound to B-lymphoma cells accumulate in shed cytoplasmic fragments

doi: 10.1038/sj.bjc.6602131

Figure Lengend Snippet: Immunoperoxidase staining of B-lymphoma cells after overnight incubation with anti-CD20 (1F5). Cells were deposited on cytocentrifuge slides, fixed with formaldehyde, permeabilised with saponin, and stained with a biotinylated horse anti-mouse IgG, followed by a complex of streptavidin and peroxidase. Observation was with an × 40 objective, except as noted. ( A ) Raji cells, showing prominent staining of JN spots. ( B ) RL cells, showing dark staining of apparently extracellular objects, referred to as CFs. ( C ) A lower-power photograph of RL cells (× 10), to show the general staining pattern. ( D ) RL cells stained in the absence of saponin, to show antigen that is accessible without permeabilisation. Control Abs of the same subclass produced no brown staining.

Article Snippet: For use with the rabbit anti-human IgM antisera, we used a biotinylated anti-rabbit IgG from Vector Labs.

Techniques: Immunoperoxidase Staining, Incubation, Staining, Produced

Journal: Human Molecular Genetics

Article Title: Human APOE genotype affects intraneuronal Aβ1–42 accumulation in a lentiviral gene transfer model

doi: 10.1093/hmg/ddt525

Figure Lengend Snippet: Intracellular accumulation of lentiviral Aβ 1–42 is greatest in APOE4-TR mice at 2 weeks after lentivirus injection. ( A ) Representative image of a coronal brain section with DAB staining of biotinylated MOAB2 for lentiviral Aβ 1–42

Article Snippet: Immediately prior to staining, brain sections from APOE2-, APOE3- and APOE4-TR mice were rinsed in 0.1 M PBS (3 × 10 min), washed in TBS (3 × 10 min), incubated in quench peroxidase (10% methanol, 3% hydrogen peroxide in 1× TBS) for 20 min, permeabilized with TBS containing 0.25%Triton X-100 (TBSX; 3 × 10 min) and blocked with 10% horse serum in TBSX for 1 h. Free-floating sections were subsequently incubated overnight at 4°C with a biotinylated anti-Aβ antibody, MOAΒ-2 (mouse, 1:1000 dilution of 0.5 mg/ml stock) or anti-tau [pSpS199/202] phospho-specific antibody (rabbit, 1:10 000 dilution), washed in TBS (3 × 10 min), incubated with biotinylated goat anti-rabbit secondary antibody for phosphorylated tau protein DAB staining (1:200) for 1 h, washed in TBS (3 × 10 min) and then incubated with avidin–biotin complex (Vector Laboratories) for 1 h. Sections were washed in TBS (2 × 15 min), rinsed in 0.1 M Tris–HCl (pH 7.5) for 3 min, and reaction products were visualized using 0.1 M Tris–HCl (pH 7.5) containing 0.05% 3/3′-diaminobenzidine tetrahydrochloride and 0.003% hydrogen peroxide.

Techniques: Mouse Assay, Injection, Staining

Journal: Biomaterials

Article Title: Enhanced biocompatibility of CD47-functionalized vascular stents

doi: 10.1016/j.biomaterials.2016.02.008

Figure Lengend Snippet: CD47 Quantification. (A) The modification chemistry summarized in was utilized to facilitate quantification. Following PEI-PDT addition, primary amines were acetylated using sulfo-NHS-Acetate. recCD47 and pepCD47 were biotinylated using Sulfo-NHS-LC-LC-Biotin.

Article Snippet: The absolute values of CD47 immobilization density were obtained by plotting the OD data generated with the sets of foil samples to a standard OD curve obtained with escalating amounts of biotinylated goat anti-rabbit antibody (Vector Lab), disclosed to contain an average of 10 biotin groups per a 150 kDa molecule.

Techniques: Modification

Journal: PLoS Biology

Article Title: Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1Rousing a Latent Defense Mechanism to Fight HIV

doi: 10.1371/journal.pbio.1000095

Figure Lengend Snippet: Expression of Retrocyclins in Cervicovaginal Tissue Model Using Aminoglycosides (A) Cervicovaginal tissues were treated with PBS (Con) or 10 μl tobramycin (Tob) and incubated with rabbit preimmune serum or antiretrocyclin antibody. The slides were then incubated with biotinylated goat anti-rabbit IgG secondary antibody and then stained using FITC-avidin. (B) Cytotoxicity was determined by measuring LDH activity in media underlying the tissues treated with PBS or tobramycin as indicated. Bars represent absorbance measured as 490 nm and error bars represent SEM; n = 6. (C) HPLC trace of extracts of tissues treated with 10 μg/ml tobramycin (tissue + Tob) and 20 μg of synthetic RC-100. (D) RC-100 synthetic peptide (indicated amounts), HPLC fractions 27–29 of control, tobramycin-treated, and RC-100 were dotted on a PVDF membrane and analyzed by immuno-dotblot.

Article Snippet: Slides were washed, incubated in biotinylated goat anti-rabbit IgG antibody (1:20,000 in 1% goat serum/PBS for 30 min), followed by additional washing and treatment with Fluorescein-Avidin D (Vector Laboratories Inc.; 1:500 in PBS for 30 min).

Techniques: Expressing, Incubation, Staining, Avidin-Biotin Assay, Activity Assay, High Performance Liquid Chromatography

Journal: PLoS Biology

Article Title: Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1Rousing a Latent Defense Mechanism to Fight HIV

doi: 10.1371/journal.pbio.1000095

Figure Lengend Snippet: Immunofluorescence Staining of Stably Transfected HL60 Cells Reveals Retrocyclin Peptides (A) Retrocyclin peptides RC-100, RC-101, and RC-101_2K peptides (in duplicates) and (B) RC-100, RC-100b, RC-101, protegrin-1 (PG-1), rhesus theta defensin-1 (RTD-1), and human neutrophil peptides 1–3 (HNP 1–3) were dotted (0–8 ng/4 μl dot) on a PVDF membrane and subjected to immuno-dotblot analysis. (C) VC, R1R3, and A1A3 (100,000 cells each) were fixed onto glass slides and incubated with rabbit anti-RC-101 antibody followed by biotinylated goat anti-rabbit IgG secondary antibody and then stained using fluorescein isothiocyanate (FITC)-avidin. Slides were visualized using Zeiss Axiovert 200M microscope system at 40× magnification. The three rows show FITC staining, DIC, and the merged image, respectively. Scale bar represents 20 μm.

Article Snippet: Slides were washed, incubated in biotinylated goat anti-rabbit IgG antibody (1:20,000 in 1% goat serum/PBS for 30 min), followed by additional washing and treatment with Fluorescein-Avidin D (Vector Laboratories Inc.; 1:500 in PBS for 30 min).

Techniques: Immunofluorescence, Staining, Stable Transfection, Transfection, Incubation, Avidin-Biotin Assay, Microscopy

Journal: Nature Communications

Article Title: Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization

doi: 10.1038/s41467-020-15987-2

Figure Lengend Snippet: 3D-EMISH method to visualize ultra-resolution 3D chromatin folding. a 3D-EMISH schematic. Cells are grown in suspension. After fixation with 4% PFA, thrombin–fibrinogen clot is formed. The clot is postfixed, soaked with cryoprotectant, frozen and cut in 40-µm sections. Free-floating sections are subjected to in situ hybridization with biotinylated DNA probe and processed to SBF-SEM. Then, multiple rounds of ultrathin slicing and imaging are performed. Each cubical sample volume contains dozens of cells. Cell nucleus is segmented, containing two separated target chromatins, as an example in 3D-EMISH. b Image processing for 3D-EMISH. First, we searched for the connected components through z -stack images per each identified nucleus (blue dotted circle). Second, the chromatin target structures were identified by removing nonspecific background EM signals. EM signals, connected in multiple consecutive layers were considered as actual target chromatin bound signals, otherwise regarded as chromatin unbound signals, or nonspecific signals. Two scale bars are 1 µm. c 3D-EMISH image example of one slice and all z -stack projected image after filtering out background EM signals. Two scale bars are 200 nm. d 3D reconstructed chromatin-folding structure examples. We assigned unique structure index number (sID) for each structure; scale bar, 500 nm.

Article Snippet: In details, after blocking of endogenous biotin (Vector Laboratories; SP-2001), the cells were permeabilized with 0.5% Triton X-100 (Sigma) in PBS (RT, 20 min).

Techniques: In Situ Hybridization, Imaging

Journal: Molecular Therapy: the Journal of the American Society of Gene Therapy

Article Title: Mimicking Aspects of Frontotemporal Lobar Degeneration and Lou Gehrig's Disease in Rats via TDP-43 Overexpression

doi: 10.1038/mt.2009.3

Figure Lengend Snippet: Labeling for apoptotic nuclei. End labeling with biotinylated nucleotides was visualized with a diaminobenzidine chromagen with a methylene green counterstain. End labeling occurred after ( a ) TDP-43 injections, but not after ( b ) control injections ( c ) higher magnification of end labeling as in a . Interval of 2 weeks and equal vector doses of 3 × 10 10 vector genomes. a,b , bar = 34 µm; c , bar = 21 µm.

Article Snippet: Biotinylated secondary antibodies for peroxidase staining were from DAKO Cytomation (1:2,000; Carpinteria, CA), incubated for 1 hour at room temperature.

Techniques: Labeling, End Labeling, Plasmid Preparation

Journal: Journal of neurochemistry

Article Title: The Orphan G Protein-coupled Receptor 75 Signaling is Activated by the Chemokine CCL5

doi: 10.1111/jnc.14463

Figure Lengend Snippet: CCL5 increases phosphorylation of signaling proteins. Differentiated SH-SY5Y cells were exposed to CCL5 (100 nM) for the indicated time points and lysates were prepared. An equal amount of proteins was loaded on a gel, transferred and immunoblotted with an antibody against ( A ) pAKT, ( C ) pGSK3β or ( E ) pERK1/2. Blots were stripped and reprobed with antibodies against AKT, GSK3β or ERK1/2. B, D and F: relative levels of phosphoproteins were calculated by optical density as described in Materials and Methods. Data are the mean + SEM of three independent samples for each time point.

Article Snippet: For assurance that an equal amount of total protein was loaded in each lane, after each experiment the membranes were stripped of the antibodies by using the Restore Western Blot Stripping Buffer (cat# 21059; Thermo Fisher Scientific) and re-probed with AKT (cat# 9272, RRID:AB_329827), ERK1/2 (cat# 5013S, RRID:AB_10693607) or GSK3β (cat# 9332s, RRID:AB_2335664) antibodies (all at 1:1000 dilution, Cell Signaling Technology).

Techniques:

Journal: Journal of Clinical Investigation

Article Title: Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF

doi:

Figure Lengend Snippet: Adverse effects of neutralization of endogenous HGF on the ischemia/reperfusion injury model. ( a ) Specificity of the neutralizing antibody to HGF. Plasma from a rat with ischemia/reperfusion injury was immunoprecipitated with normal IgG (lane 1) or anti–rat HGF IgG (lane 2), and immunoreactive proteins were detected by Western blot, using biotinylated anti–rat HGF IgG. ( b ) Immunohistochemical staining of infarcted hearts with α-sarcomeric actin to depict the infarct area and its quantification. Anti–rat HGF IgG ( n = 10) or normal IgG ( n = 10) was injected 20 minutes before coronary occlusion, and every 12 hours after reperfusion. Forty-eight hours after operation, rats were killed and histological and biochemical analyses were made. Arrowheads indicate the α-sarcomeric actin–negative infarct area (original magnification, ×40). A P

Article Snippet: After blocking, the membrane was sequentially incubated with anti–phospho-p44/p42 mitogen-activated protein kinase (ERK-1/2) antibody (E10; New England BioLabs Inc., Beverly, Massachusetts, USA), biotinylated anti-mouse IgG (Vector Laboratories), horseradish peroxidase–conjugated streptavidin (Amersham Pharmacia Biotech UK, Little Chalfont, United Kingdom), and an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech UK).

Techniques: Neutralization, Immunoprecipitation, Western Blot, Immunohistochemistry, Staining, Injection

Journal: International Journal of Molecular Sciences

Article Title: MUC16/CA125 in the Context of Modular Proteins with an Annotated Role in Adhesion-Related Processes: In Silico Analysis

doi: 10.3390/ijms130810387

Figure Lengend Snippet: CA125-immunoreactivity of Herpesvirus antigens. Mouse monoclonal anti-human CA125 antibodies: clone X306 (OC125-like) and clone X325 (M-11 like) were allowed to react with immobilized Epstein-Barr Virus (EBV) capsid antigens or Herpes simplex virus type 1 antigens (HSV1). Binding was detected using biotinylated goat anti-mouse IgG and Vectastain Elite ABC reagent. The absorbance was measured at 450 nm. Non-specific binding was estimated using an irrelevant monoclonal anti-hCG antibody (c).

Article Snippet: After incubation for 3 h at room temperature (RT), the wells were washed three times with 0.1 M PBS, pH 7.2 and biotinylated goat anti-mouse IgG (Vector Laboratories, Burlinghame, CA, USA) was added.

Techniques: Binding Assay

Journal: The Journal of Cell Biology

Article Title: Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration

doi: 10.1083/jcb.201110032

Figure Lengend Snippet: SSPN increases cell surface glycosylation in mdx muscle. (A and B) Transverse cryosections of quadriceps muscles from SSPN-Tg (A) or Akt transgenic (B) muscles were stained with biotinylated Wisteria floribunda agglutinin (WFA) and visualized by indirect immunofluorescence. Bars, 50 µm. (C) Skeletal muscle protein lysates from the indicated mouse models were enriched by WFA lectin affinity chromatography (WFA enrichment) and analyzed with indicated antibodies and overlayed (O/L) with WFA lectin (WFA O/L). (D) Skeletal muscle protein lysates were enriched by WFA lectin affinity chromatography (WFA enrichment) and subjected to the same analysis as described in C. (E) WFA and laminin overlay assays were performed on protein lysates enriched with succinylated WGA (sWGA) lectin chromatography from mdx and Akt transgenic mdx ( mdx Akt ) muscle. Mice were treated with doxycycline to induce Akt expression in skeletal muscle as described previously ( Peter et al., 2009 ). Laminin overlays (Lam O/L) represent binding to immobilized α-DG on nitrocellulose transfers. Immunoblotting with antibodies to α-DG is shown. (F) Levels of WFA binding to α-DG were quantitated by densitometry of bands from overlay assays, and data are expressed relative to mdx levels (100%). Error bars represent standard deviation of the mean ( n = 2–3 muscle preps per genotype). (G) Quantitative RT-PCR was used to investigate whether overexpression of SSPN alters RNA levels of CT GalNAc transferase ( Galgt2 ). RNA was isolated from WT, WT 1.5 , mdx , and mdx 1.5 skeletal muscle. Data are expressed relative to non-Tg WT controls. Error bars represent standard error of the mean (*, P

Article Snippet: Primary antibodies were detected by biotinylated anti–rabbit (BA-1000; 1:500; Vector Laboratories) and biotinylated anti–mouse (BA-9200; 1:500; Vector Laboratories).

Techniques: Transgenic Assay, Staining, Immunofluorescence, Affinity Chromatography, Whole Genome Amplification, Chromatography, Mouse Assay, Expressing, Laser Capture Microdissection, Binding Assay, Standard Deviation, Quantitative RT-PCR, Over Expression, Isolation

Journal: The Journal of Cell Biology

Article Title: Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration

doi: 10.1083/jcb.201110032

Figure Lengend Snippet: SSPN regulates utrophin levels and glycosylation of α-DG in myd mice. (A) Transverse cryosections of quadriceps muscles were stained with the indicated antibodies and overlayed with biotinylated WFA. (B) Transverse cryosections of quadriceps muscles were stained with indicated antibodies and overlayed with biotinylated WFA. Staining with IIH6 antibodies, which recognize LARGE epitopes on α-DG, was not detected in myd samples, as expected ( Fig. S4 ). Transverse cryosections of skeletal muscle from 4–6-wk-old myd , SSPN-Tg: myd ( myd 3.0 ), and SSPN-deficient myd ( myd :SSPN −/− ) mice were stained with H E. Muscle sections were stained with antibodies to embryonic myosin heavy chain (eMHC; green) as a marker for newly regenerated myofibers. Mice were injected with Evans blue dye (EBD), a marker for membrane instability (visualized by red fluorescence). Sections were costained with laminin antibodies (green fluorescence) to visualize individual fibers. (C) Quantification of central nucleation, Evans blue dye–positive fibers, and eMHC-positive fibers is expressed as a percentage of total fibers. Error bars represent standard deviation of the mean ( n = 4 quadriceps per genotype). (D–G) Skeletal muscles from the indicated mice were enriched using either sWGA or WFA lectin chromatography. Immunoblots of 10-µg protein eluates are shown. A.U., arbitrary unit; Utr, utrophin. Bars, 50 µm.

Article Snippet: Primary antibodies were detected by biotinylated anti–rabbit (BA-1000; 1:500; Vector Laboratories) and biotinylated anti–mouse (BA-9200; 1:500; Vector Laboratories).

Techniques: Mouse Assay, Staining, Marker, Injection, Fluorescence, Standard Deviation, Chromatography, Western Blot

Journal: The American Journal of Pathology

Article Title: Fibroblast Heterogeneity

doi:

Figure Lengend Snippet: Immunohistochemical localization of Thy 1 in human myometrium and endometrium. Frozen tissue sections were fixed in formalin and blocked with 5% normal horse serum for 20 minutes at room temperature. Tissue sections were incubated overnight at 4°C with mouse monoclonal anti-human Thy 1 (F15-421-5, 10 μg/ml). A negative control antibody (mIgG1) was included at 10 μg/ml. Sections were incubated with biotinylated horse anti-mouse IgG and the avidin-biotin-peroxidase detection system was used. Sections were counterstained with hematoxylin. a: Myometrium. Immunoreactivity is present in some smooth muscle cells and in some fibroblastic stromal cells. b: Myometrium. Negative control; primary antibody replaced with mouse immunoglobulin. c: Endometrium basalis. Thy 1 is localized mainly to the perivascular region. d: Endometrium basalis. Negative control. e: Endometrium functionalis. Thy 1 immunoreactivity is present in some stromal cells. f: Endometrium functionalis. Negative control. Original magnifications, ×400. Scale bar, 50 μm.

Article Snippet: Sections were then incubated with biotinylated horse anti-mouse IgG (Vector Laboratories) and the avidin-biotin-peroxidase detection system was used (Elite ABC 6101; Vector Laboratories).

Techniques: Immunohistochemistry, Incubation, Negative Control, Avidin-Biotin Assay

Journal: The Journal of Experimental Medicine

Article Title: BCMA Is Essential for the Survival of Long-lived Bone Marrow Plasma Cells

doi: 10.1084/jem.20031330

Figure Lengend Snippet: (A) PCs express BLyS receptor. Immune BM and spleen (SP) was isolated and stained with αCD138, αB220, and biotinylated BLyS. Histograms of BLyS receptor expression are shown for B220 + CD138 − cells (red); B220 − CD138 − cells (green); and B220 − CD138 + cells (blue). Data are representative of three independent experiments. (B) BM PCs express heightened levels of BCMA mRNA but not BAFF-R or TACI. RNA from splenic B cells (B cells), αCD40-induced B cell blasts (αCD40 B), or BM PCs (PC) was isolated via the TRIzol method. Real-time RT-PCR was performed with each sample to evaluate the relative levels of mRNA expression for BCMA, TACI, and BAFF-R. Data are representative of three experiments. (C) BLyS enhances Mcl-1 expression in PCs but not B cells. Purified CD138 + BM PCs or splenic B cells were cultured with the indicated stimulus, as described in Fig. 1 . After 18 h, RNA was isolated, and the relative expression of antiapoptotic genes was determined relative to β-actin.

Article Snippet: Incubation with biotinylated antibodies was followed by incubation with streptavidin, peridinine chlorophyll protein, or CyChrome or allophycocyanin (BD Biosciences).

Techniques: Isolation, Staining, Expressing, Quantitative RT-PCR, Purification, Cell Culture

Journal: Current Alzheimer Research

Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

doi: 10.2174/1567205015666180801111033

Figure Lengend Snippet: Both the isotype control and anti-CD49d antibody injections affected brain CD4 immunoreactivity. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. The presence of IgG2b antibody in the brain was assessed by immunostaining using biotinylated anti-mouse IgG2b antibody (8A) . The presence of T cells in the brain was visualized using anti-CD4 antibody and immunoreactivity observed in the striatum (8B) and parietal cortex (8C) was imaged. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets.

Article Snippet: Elite Vectastain ABC avidin and biotin kits, biotinylated anti-rabbit, anti-mouse, and anti-rat antibodies and the Vector VIP kits were from Vector Laboratories Inc. (Burlingame, CA).

Techniques: Mouse Assay, Injection, Purification, Immunostaining

Journal: Journal of neuroscience methods

Article Title: Fluorescent Arc/Arg3.1 indicator mice: a versatile tool to study brain activity changes in vitro and in vivo

doi: 10.1016/j.jneumeth.2009.07.015

Figure Lengend Snippet: Increased d4EGFP expression in a mouse model of Alzheimer’s disease Representative images of DG hippocampus (coronal sections) from one month-old Tg Arc/Arg3.1-d4EGFP and Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP littermate mice are shown after visualization of d4EGFP by DAB (A, B) or immunofluorescence (C,D). Note the increased number of and labeling intensity of d4EGFP-positive neurons in CA1 (B) and DG granule cells (B, D) in the Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice. Brain sections stained with anti-GFP antibody and visualized by biotinylated (DAB method) or FITC-conjugated secondary antibodies. The fluorescent images are single confocal scans. Abbreviations: db – dorsal blade of DG, vb – ventral blade of DG; scale bars = 250 μm (A,B) and 100 μm (C,D). (E) Count of d4EGFP-positive neurons in Tg 5XFAD /Tg Arc/Arg3.1-d4EGFP mice, as percent change from control Tg Arc/Arg3.1-d4EGFP littermates, in the DG: (% mean ± SD): 216.3 ± 7.7 vs. 100 ± 6.4 (**p

Article Snippet: Briefly, slices were blocked with 10% NGS containing 0.1% Triton X-100 antibody in PBS for 1 hr and reacted with either anti-Arc (Arg3.1 antiserum, provided by Dietmar Kuhl, dilution 1:1,500) or anti-GFP (Molecular Probes, Cat# , dilution 1:2,000) rabbit antibody in 1% NGS containing PBS overnight at 4 C. After reaction with biotinylated anti-rabbit antibody (Vector laboratory, dilution 1:200), immunoreactions were visualized with the ABC elite kit (Vector laboratory) and DAB development procedure.

Techniques: Expressing, Mouse Assay, Immunofluorescence, Labeling, Staining

Journal: Frontiers in Neural Circuits

Article Title: Rat whisker motor cortex is subdivided into sensory-input and motor-output areas

doi: 10.3389/fncir.2013.00004

Figure Lengend Snippet: Corticocortical projections from SI barrel cortex terminate at MI sites located lateral to the sites most effective for evoking whisker movements. (A) Tangential section processed for cytochrome oxidase (CO) shows the spatial distribution of the layer IV barrels in SI cortex. (A′) An adjacent section processed for biotinylated dextran amine (BDA) shows the location of BDA deposits in SI barrel cortex. Contour lines indicate the primary (SI) and secondary (SII) somatosensory cortical areas as well as the posterior parietal cortex (PPC). Rectangle indicates the region depicted in panel (B) . (B) Location of two electrolytic lesions (red arrows) marking where intracranial microstimulation (ICMS) was most effective in evoking whisker twitches. Labeled projections (arrowheads) from SI terminate in a strip of MI cortex located caudal and lateral to sites that evoked the best whisker responses. Scale bars: 2 mm in (A) ; 500 μm in (B) .

Article Snippet: The tracers were either a 15% solution of biotinylated dextran amine (BDA, Invitrogen) or a 15% solution of Fluoro-Ruby (FR, Invitrogen).

Techniques: Whisker Assay, Labeling, Stripping Membranes

Journal: Immunology

Article Title: Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1

doi: 10.1111/imm.12229

Figure Lengend Snippet: Ustekinumab inhibits CXCL12-induced T-lymphocyte motility and effects on lipoprotein receptor-related protein 1 (LRP1) and thrombospondin 1 (TSP-1). (a) The influence of ustekinumab (100 μg/ml) on migration into a collagen matrix in the absence and presence of CXCL12 (50 ng/ml) as determined after 30 min. (b) Gel analysis [4–12% SDS–PAGE (LRP1) and 6% SDS–PAGE (TSP-1)] showing the influence of ustekinumab, CXCL12 and dynasore on the cell surface expression of TSP-1 and LRP1. The cells were surface biotinylated and immunoprecipitated after 10 min. (c) Western blotting (6% SDS–PAGE) of material from lysed cells after incubation for 30 min with and without CXCL12 (50 ng/ml). (d) Western blotting of material from lysed cells using different anti-TSP-1 antibodies (Ab9 reacts with the N-terminal domain of TSP-1) after incubation for 30 min with CXCL12 (50 ng/ml). The results in (a) show mean values of three independent experiments. The results in (b–d) show one representative experiment of three to seven independent experiments.

Article Snippet: Biotinylated peroxidase and avidin were from Vector Laboratories (Burlingame, CA).

Techniques: Migration, SDS Page, Expressing, Immunoprecipitation, Western Blot, Incubation

Journal: Immunology

Article Title: Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1

doi: 10.1111/imm.12229

Figure Lengend Snippet: Ustekinumab inhibits T-lymphocyte motility through lipoprotein receptor-related protein 1 (LRP1) and thrombospondin 1 (TSP-1). (a,b) Motility was examined with cells in the presence of ustekinumab (100 μg/ml) or infliximab (100 μg/ml on intercellular adhesion molecule 1 (10 μg/ml) (a) and fibronectin (10 μg/ml) (b) as determined by a transwell assay measuring number of transmigrated cells. (c) Formation of conjugates of AF24 T cells with antigen-presenting HLA-identical B cells in the presence of ustekinumab (100 μg/ml) or infliximab (100 μg/ml). (d) Gel analysis (6% SDS–PAGE) showing the influence of incubation with ustekinumab on the cell surface expression of TSP-1, LRP1 and CD4 after 15 min, as demonstrated by immunoprecipitation of surface biotinylated cells. (e) Quantitative immunocytochemistry showing the influence of ustekinumab (100 μg/ml) and infliximab (100 μg/ml) on cell-surface-expressed and intracellular LRP1 and TSP-1 as determined after 30 min. The cell surface expression of CD4 is shown as a comparison. (a,b) Mean values of three independent experiments and (c) one representative experiment of two independent experiments. The results in (d) and (e) show one representative experiment of three to five independent experiments.

Article Snippet: Biotinylated peroxidase and avidin were from Vector Laboratories (Burlingame, CA).

Techniques: Transwell Assay, SDS Page, Incubation, Expressing, Immunoprecipitation, Immunocytochemistry

Journal: The Journal of Neuroscience

Article Title: Assembly with the NR1 Subunit Is Required for Surface Expression of NR3A-Containing NMDA Receptors

doi: 10.1523/JNEUROSCI.21-04-01228.2001

Figure Lengend Snippet: NR3A is present at the cell surface only when coexpressed with the NR1-1a subunit. A , HEK293T cells were transfected with different combinations of NMDA receptor subunits and incubated for 15 min with sulfo-NHS-biotin. After solubilization, biotinylated protein was recovered by streptavidin precipitation. The streptavidin fractions ( lanes labeled 2 ), representing the membrane proteins, and aliquots of the lysate before ( lanes labeled 1 ) and after ( lanes labeled 3 ) streptavidin precipitation were analyzed by immunoblotting using anti-NR1, anti-NR2A/B, anti-NR3A, and anti-calreticulin antibodies. An excess amount of protein was loaded in the lanes labeled 2 to ensure detection of any NR3A or calreticulin at the cell surface. The subunit combinations used for transfection are indicated above each blot, and the positions of molecular size markers in kilodaltons are shown on the left . A representative experiment is shown; n = 3. B, C , Surface localization of GFP-tagged NR3A. B, Left , Schematic drawing of expected transmembrane ( TM ) topology of NR3A-GFP is shown. Right , Protein immunoblots of HEK293T cells transfected with NR3A or NR3AGFP and probed with anti-NR3A antibody show an increase in NR3A molecular weight that corresponds to the molecular mass of GFP (27 kDa). No lower molecular weight bands were observed. C , Cells transfected with GFP-tagged NR3A alone or in combination with the other NMDA receptor subunits were immunostained in nonpermeabilizing (NP) conditions with anti-GFP antibody followed by a Texas Red-conjugated secondary antibody and imaged with filters for GFP and Texas Red. All four panels show raw superimposed confocal images combining NP anti-GFP antibody staining ( red ) and native GFP fluorescence from NR3A-GFP ( green ). Yellow corresponds to the overlap of GFP immunostaining and GFP fluorescence and reflects NR3A-GFP expressed at the cell surface. Because the intensity of red immunostaining was brighter than was green GFP fluorescence, regions of overlapping can appear red-yellow . When expressed alone, NR3A-GFP exhibits a perinuclear and reticular fluorescence pattern, and no surface staining is observed. Cotransfection of NR1-1a/NR2A leads to the appearance of patches of fluorescence at the plasma membrane. Scale bar, 10 μm.

Article Snippet: Biotinylated anti-rabbit secondary antibody (1:1000; Vector Laboratories) followed by avidin-Texas Red (1:1000; Vector Laboratories) or Cy3-conjugated anti-mouse antibody (1:600; Jackson ImmunoResearch) was used.

Techniques: Transfection, Incubation, Labeling, Western Blot, Molecular Weight, Staining, Fluorescence, Immunostaining, Cotransfection