Fresh frozen slice culture tissues were homogenized in NP-40 lysis buffer supplemented with protease and phosphatase inhibitors [36 (link)]. Protein concentrations were determined with the bi cinchoninic acid (BCA) assay (Pierce, Rockford, IL). Immunoreactivity was measured in 4 replicate 100 ng protein samples by direct binding ELISA [36 (link)], and protein loading was subsequently quantified by measuring immune reactivity to large acidic ribosomal protein (RPLPO) [36 (link)]. Primary antibodies were diluted to 0.1-0.5 pg/ml, and their binding was detected with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000; Pierce, Rockford, IL) and Amplex Ultra Red soluble fluorophore (Molecular Probes, Eugene, OR). Amplex Red fluorescence fluorescent light units (FLU) were measured in a SpectraMax M5 (Ex 530/Em 590). Subsequently, the samples were incubated with biotin-conjugated polyclonal antibodies to RPLPO, and immunoreactivity was detected with streptavidin-conjugated alkaline phosphatase (1:1000; Vector, Burlingame, CA) and the 4-Methylumbelliferyl phosphate (4-MUP) fluorophore (Molecular Probes, Eugene, OR) (Ex360/Em450). Binding specificity was assessed with negative control incubations in which the primary or secondary antibody was omitted. The calculated ratios of specific protein/RPLPO fluorescence were used for inter-group statistical comparisons.
Alkaline phosphatase conjugated streptavidin
Manufactured by Vector Laboratories
Sourced in United States, Canada
Alkaline phosphatase-conjugated streptavidin is a protein complex used as a detection reagent in various biotechnical applications. Streptavidin, a protein derived from the bacterium Streptomyces avidinii, is covalently linked to the enzyme alkaline phosphatase. This conjugate retains the high-affinity binding properties of streptavidin towards biotin, a small molecule often used to label biological targets.
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Lab products found in correlation
Vector red kit, by Vector Laboratories (3 mentions)
Cd34 microbead kit ultrapure, by Miltenyi Biotec (1 mentions)
Evans blue, by Fujifilm (1 mentions)
Human il 6 hil 6, by Thermo Fisher Scientific (1 mentions)
Biotin conjugated anti mouse igg, by Agilent Technologies (1 mentions)
Vector red substrate, by Vector Laboratories (1 mentions)
Hrp anti mouse, by Agilent Technologies (1 mentions)
Antigen retrieval solution, by Agilent Technologies (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Peroxidase blocking solution, by Agilent Technologies (1 mentions)
Permount, by Vector Laboratories (1 mentions)
Streptavidin biotin blocking solution, by Vector Laboratories (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Methyl green, by Agilent Technologies (1 mentions)
Methyl green, by Vector Laboratories (1 mentions)
8 protocols using alkaline phosphatase conjugated streptavidin
1
Quantitative Protein Measurement in Tissue Samples
2
Megakaryocyte Colony Formation Assay
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A Mk colony formation assay was performed as described previously33 (link),34 (link) with minor modifications. CD34-positive cells were purified from cryopreserved bone marrow mononuclear cells (BM-MNCs) using CD34 MicroBead Kit UltraPure (MACS Miltenyi Biotec). CD34-positive cells (5000) were cultured in a chamber (two-chamber slide, Matsunami) with serum-free collagen medium in the presence of 20 ng/mL human IL-3 (hIL-3) (Miltenyi Biotec) and human IL-6 (hIL-6) (Peprotech) and in the presence or absence of 50 ng/mL human thrombopoietin (hTPO) (Kyowa Kirin). The cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air for 12–14 days. Fixed cells were stained with anti-CD41 antibody (Clone HIP8, STEMCELL), biotin-conjugated anti-mouse IgG (Dako), streptavidin-conjugated alkaline phosphatase (Vector Laboratory), SIGMA FAST Red TR/naphthol AS-MX alkaline phosphatase substrate (Sigma), and Evans Blue (FUJIFILM Wako). BM-MNCs collected from lymphoma patients without BM infiltration and reactive thrombocytosis patients were used as negative controls.
3
Quantitative Protein Measurement in Tissue Samples
4
Immunohistochemical Analysis of Myostatin
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Paraffin sections (5 μm) of 2% paraformaldehyde-fixed tissues were deparaffined, hydrated and subjected to antigen-retrieval (microwave oven treatment in 0.1 M sodium citrate). Staining was performed after quenching of endogenous peroxidase with 3% H2O2 in methanol. Slides were incubated with primary antibody overnight, followed by incubation with biotinylated antibody for 30 minutes. Each sample was analyzed for the detection of Mstn (mouse monoclonal ab, biorbyt, Cambridge, UK) and a labelled polymer HRP anti-mouse from Dako was used as secondary antibody. Staining with CD45 (Novocastra, Leica Microsystem, Milano Italy), α-SMA (Dako Italia s.r.l, Agilent Pathology Solution, Cernusco sul Naviglio, Italy) were completed with the appropriate secondary antibody using the streptavidin-peroxidase method, performed as previously described55 (link). The expression of Mstn was examined by image analysis and expressed as positive areas. In order to evaluate the co-distribution of two different antigens in the same sample, a double immunohistochemistry procedure was carried out. First, one antibody was evidenced by streptavidin-peroxidase and the second by alkaline phosphatase-conjugated streptavidin (Vector Laboratories, CA, USA). The alkaline phosphatase substrate was Vector ®RED Substrate (Vector Laboratories).
5
Immunohistochemical Analysis of Tissue Samples
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All tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin, and sections (5-µm thickness) were stained with hematoxylin and eosin or processed for antibody (Ab) staining. For IHC staining, sections were deparaffinized and treated with an antigen-retrieval solution (Dako, Carpinteria, CA) at 98°C for 25 min, a peroxidase-blocking solution (Dako) for 15 min, and then with a streptavidin-biotin blocking solution (Vector Laboratories, Burlingame, CA) for 1 h. Sections were incubated for 2 h at room temperature with one of the following antibodies: rabbit anti-O. tsutsugamushi Karp strain polyclonal antibody (pAb, 1∶500); rat anti-mouse neutrophil monoclonal antibody (mAb, 1∶25, clone 7/4, Caltag Laboratories, Buckingham, UK); rabbit anti- myeloperoxidase (MPO) pAb (1∶25, Abcam); or rabbit anti-mouse CD3 mAb (1∶25, Abcam, Cambridge, MA). Biotinylated goat anti-rat or anti-rabbit secondary antibodies (1∶200, Vector) were incubated on the sections for 30 min. Sections were stained with alkaline phosphatase-conjugated streptavidin (1∶200, Vector), Vector Red alkaline phosphatase substrate, and counterstained with hematoxylin (Sigma, St. Louis, MO). Reagent negative controls consisted of samples in which primary Ab was replaced with normal rat or rabbit IgG. Sections were dehydrated, mounted in Permount (Vector), and imaged under an Olympus BX53 microscope.
6
Assessing Ameloblast Barrier Function
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The apical tight junction is an important indicator for ameloblast polarity and can be measured by the functional selectivity of the paracellular apical barrier. Sulfo-NHS-Biotin was used as a tracer molecule to evaluate the effects of SATB1 on the ameloblast layer barrier function as previously described [78 (link)]. Briefly, four 10-day old pups from each wt and Satb1−/− mouse model were anesthetized by intraperitoneal injection with 0.05 ml/10 g of ketamine (18 mg/ml). A 30-G injection needle with a blunt end was carefully inserted into the left ventricle of the heart, and 150 μl/g of Sulfo-NHS-Biotin solution was perfused into the bloodstream. Ten minutes after receiving Sulfo-NHS-Biotin injection, the pups were sacrificed, and the hemimandibles were dissected, processed, and paraffin-embedded for histological sectioning as previously mentioned. The sagittal sections were blocked with 5% BSA for 1 h then incubated with alkaline phosphatase-conjugated streptavidin (Vector Laboratories, Inc.) for 30 min, and immunoreactivity was visualized using a Vector® Red kit (Vector Laboratories, Inc.).
7
Mandible Immunohistochemistry for AR, TGFBR2, TGFB1, PR
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Mouse mandibles were fixed in 4% paraformaldehyde in 0.06 M sodium cacodylate buffer (pH 7.3) at 4°C for 24 h. After decalcification in 8% EDTA (pH 7.3), samples were processed for routine paraffin embedding and sagittally sectioned. The sections were incubated with 10% swine and 5% goat sera followed by incubation with rabbit anti-human AR (1:75; Novus Biologicals, Littleton, CO, NB100-91658), rabbit anti-mouse TGFBR2 (1:100; Santa Cruz Biotech, Santa Cruz, CA, sc-1700), rabbit anti-human TGFB1 (1:50; Abcam PLC, Cambridge, MA, ab92486), and rabbit anti-mouse PR (Santa Cruz Biotech, sc-166170) antibodies respectively overnight at room temperature. A biotinylated swine anti-rabbit IgG F(ab')2 fraction (Dako, Carpinteria, CA) was used as the secondary antibody for 1 h at room temperature incubation. Following incubation with alkaline phosphatase conjugated streptavidin (Vector Laboratories Inc., Burlingame, CA) for 30 min, immunoreactivity was visualized using a Vector® Red kit (Vector Laboratories) resulting in pink/red color for positive staining. Counter-staining was performed with methyl green (Dako). Negative control was done with normal rabbit sera.
8
Immunohistochemical Analysis of Mandible
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Dissected mandibles were immediately immersed into 4% PFA for 1 day at 4°C followed by decalcification in 8% EDTA (pH 7.3) at 4°C for 4 weeks. The mandibles were then processed through a graded series of ethanol and xylene followed by routine embedding in paraffin and sectioning. The sections were deparaffinized, and blocked for nonspecific staining with 10% swine and 5% goat sera for 1 hour, followed by incubation with rabbit anti-mouse SATB1 antibody (Epitomics, Burlingame, CA) or rabbit anti-phosphorylated PKCα antibody (Abcam, Cambridge, MA) overnight at 4°C. The sections were next thoroughly washed with 0.2% Tween/PBS, and incubated with a biotin-conjugated swine anti-rabbit IgG F(ab’)2 fraction (Dako Cytomation Inc., Carpinteria, CA) for 1 hour at room temperature. The sections were then incubated with alkaline phosphatase conjugated streptavidin (Vector Laboratories Inc., Burlingame, CA) for 30 minutes, and immunoreactivity was visualized using a Vector Red kit (Vector Laboratories Inc.) resulting in pink/red color for positive staining. Counter-staining was done with methyl green (Vector Laboratories Inc). The sections were photographed with a Nikon Eclipse 300 microscope (Compix Inc, Sewickley, PA).
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