Cells fixed, permeabilized and immunolabelled for indicated proteins were crosslinked with 4% PFA in DPBS for 20 min and incubated with 1 mg per mL RNase (VWR Life Science, 866) in DPBS for 20 min at 37 °C. Cells were then washed twice in DPBS, dehydrated in 70%, 85%, and 100% EtOH and air-dried before the DNA was denatured at 80 °C in hybridization buffer (10 mM NaHPO4 (pH 7.4), 10 mM NaCl, 20 mM Tris (pH 7.5), 70% formamide and 1% maleic acid buffer (100 mM Maleic acid, 150 mM NaCl, 175 mM NaOH, pH 7.2) with 100 mg per mL blocking reagent (Sigma-Aldrich, 11096176001) containing 800 ng per mL TelC-Alexa488 PNA telomere probe (Panagene, F1004) for 4 min, and cells were incubated for 2 h at 22 °C in a humid and dark chamber. After 10 min wash in DPBS with 0.1% Tween-20 (65 °C) and 1 min wash in 2x SCC buffer (0.3 M NaCl, 30 mM sodium citrate, pH 7.2) with 20% Tween-20, the cells were dehydrated in 70%, 85%, and 100% EtOH and air-dried before the DNA was stained with 2.5 µg per mL Hoechst 33342. The coverslips were mounted on glass slides using Prolong Gold Antifade mounting medium before confocal microscopy.
Blocking reagent
Manufactured by Merck Group
Sourced in United States, Macao
Blocking reagent is a laboratory product used to prevent non-specific binding in various assay techniques, such as Western blotting, ELISA, and immunohistochemistry. It is designed to block unoccupied binding sites on a solid support, reducing the likelihood of unwanted interactions between the sample and the assay components.
Automatically generated - may contain errors
Lab products found in correlation
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Bcip nbt color development substrate, by Promega (2 mentions)
Heparin, by Merck Group (2 mentions)
Chaps, by Merck Group (2 mentions)
Formamide, by Avantor (2 mentions)
Ambion maxiscript t7 transcription kit, by Thermo Fisher Scientific (2 mentions)
Anti dig alkaline phosphatase conjugate, by Roche (2 mentions)
Pd 10 desalting column, by GE Healthcare (2 mentions)
Bcip nbt, by Merck Group (2 mentions)
Pcr dig labeling mix, by Roche (2 mentions)
Nbt bcip, by Roche (2 mentions)
Anti dig ap, by Merck Group (2 mentions)
Spei hf, by New England Biolabs (2 mentions)
Mlui hf, by New England Biolabs (2 mentions)
Poly l lysine solution, by Merck Group (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Metal enhanced dab kit, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
19 protocols using blocking reagent
1
Telomere Length Quantification by PNA FISH
2
Cell-Cell Adhesion Mediated by Click Chemistry
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Flow chambers were prepared by gluing coverslips to glass slides with holed parafilm. As mentioned above, the Mal-Cys -treated GFP+ HEK 293T cells were incubated with 100 μM TCEP (or without for the control group) at 37 °C for 30 min to expose the –SH groups on their surfaces. For the adhesion of the Mal-CBT -treated BFP+ HEK 293T cells to the coverslip, chambers were pre-absorbed with poly-l -lysine solution (0.01%, Sigma) for 10 min at RT (25–26 °C). Then the chambers were washed with PBS of 10-fold the chamber volume, and were incubated with BFP+ cells for 20 min. After removing the unbound BFP+ cells, the GFP+ cells with a blocking reagent at low concentration (0.06% casein, Sigma) were introduced into the chamber. The blocking reagent could prevent the adhesion of the GFP+ cells to the coverslip and lessen the non-specific interactions between the GFP+ and BFP+ cells, but could not interfere with the click reactions between the GFP+ and BFP+ cells.
3
Quantifying Macrophage and Neutrophil Accumulation
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Paraformaldehyde-fixed cryosections (5 μm thick) were treated with 3% hydrogen peroxide, avidin-biotin blocking solution, 0.5% blocking reagent (Sigma), then incubated with rat anti-mouse F4/80 (1:200) or rat anti-mouse Ly-6B.2 (7/4, 1:200), and subsequently with biotin-labeled secondary antibodies.[28 (link), 32 (link)] Immunosignals were detected using a VECTASTAIN Elite ABC kit (Vector Laboratories) and a metal-enhanced DAB kit (Thermo Fisher Scientific), counterstained with hematoxylin, and observed under light microscope (Olympus BX51). For quantitative evaluation of macrophage and neutrophil accumulation, the numbers of F4/80-positive and Ly-6B.2-positive cells were counted in 10 randomly selected high-power fields (× 200). [28 (link)]
4
Inhibition of Cellular Pathways
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NDGA (Sigma), C646 (Sigma), A485 (Tocris), and Bafilomycin A1 (Adipogen) were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich), Chloroquine (Biovision) was dissolved in water. Phosphate-buffered saline (PBS) was purchased from Corning. Tris-buffered saline with Tween (TBST) buffer (150 mM NaCl, 0.01% (v/v) Tween-20, 50 mM Tris-HCl buffer, pH 7.6) was used for immunoblotting. Blocking buffer and primary antibody incubation solution was 5% (w/v) BSA (Sigma), pH 7.0, in TBST. Secondary antibody incubation buffer was 5% (w/v) blocking reagent (Sigma) in TBST.
5
Immunohistochemistry for Kinetochore and Histone Modifications
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To detect kinetochore proteins or histone modifications, immunohistochemistry was conducted after RGEN-ISL. The primary antibody solutions consisted of primary antibody solutions diluted 1:100 in a blocking solution [100 mM Tris–HCl, 150 mM NaCl, and 0.5% (w/v) blocking reagent (Sigma-Aldrich, St. Louis, MO, USA)]: anti-OsCENH3 rabbit antibody (Nagaki et al., 2004 (link)) for centromere-specific histone H3 variant (CENH3) in rice or anti-GmCENH3 rabbit antibody (Tek et al., 2010 (link)) for CENH3 in soybean and common bean, and anti-H3K9me2 mouse antibody (MABI0317; MBL, Nagoya, Japan). After post-fixing and washing with PBS, the tissues were incubated in the primary antibody solutions at 4 °C for 12–16 h and then washed twice in PBS for 10 min at room temperature. The secondary antibodies used were Alexa Fluor 647-labeled anti-rabbit antibodies (Molecular Probes, Eugene, OR, USA) and Alexa Fluor 488-labeled anti-mouse antibodies (Molecular Probes). Both antibodies were diluted 1:500 with the blocking solution, reacted at 37 °C for 1 h, and then washed in the same way as for the primary antibodies. The tissues were transferred on to glass slides and mounted as above.
6
In Situ Hybridization for Murine BMP4
7
In Situ Hybridization for Murine BMP4
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Probe preparation and in situ hybridization were carried out as described previously with several modifications 21 (link), 22 (link). In brief, antisense DIG (digoxigenin)-labelled RNA probes were transcribed in vitro from the plasmid with the cDNA encoding of murine BMP4 using the Ambion® MAXIscript® T7 Transcription Kit (Ambion, Waltham, MA, USA). E13.5 embryos were fixed with 4% PFA for overnight then embedded in Tissue-Tek OCT compound (Sakura Finetek). Ten μm-thick cryosections were treated with proteinase K, and then prehybridized in prehybridization buffer containing 50% formamide (Amresco, Solon, OH), 5× SSC (Invitrogen, USA), 50 µg/mL tRNA (Roche, Mannheim, Germany), 50 µg/mL heparin (Sigma-Aldrich, St. Louis, MO), 2% blocking reagent (Sigma-Aldrich, St. Louis, MO), 0.1% CHAPS (Sigma-Aldrich, St. Louis, MO), 0.1% Tween 20 and 1 mM EDTA at 70°C for 2 h. Hybridization was performed with DIG-labeled RNA probes for 16 hours. After incubating with anti-DIG alkaline phosphatase conjugate (Roche Diagnostics USA) overnight, hybridization signal was visualized by using the BCIP/NBT color development substrate (Promega, Madison, WI).
8
In Situ Hybridization of CSpV1-dsRNAs
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DIG-labeled antisense CSpV1-dsRNAs were synthesized employing SP6/T7 Transcription Kit (Sigma-Aldrich, catalogue no. 10999644001) as described above. Cells were incubated and treated on Nunc™ Lab-Tek™ II Chamber Slide™ System (Thermo Scientific, catalogue no. 154534). The cells were fixed in 4% paraformaldehyde (Thermo Fisher Scientific, catalogue no. 28908) with 5% acetic acid (Fisher Scientific, catalogue no. BP2401-212) and 0.9% NaCl for 30 min, and washed twice with diethylpyrocarbonate (DEPC, Sigma-Aldrich, catalogue no. 06756) treated 1× PBS. Fixed cells were permeabilized in 0.1% Triton X-100/PBS (Bio-rad, catalogue no. 1610407) with 100 U/ml RNase inhibitor for 10 min and washed three times with 1× DEPC-PBS. Hybridization occurred in DIG Easy Hyb Granules (Sigma-Aldrich, catalogue no. 11 796 895 001) according to manufacturer’s guide. Blocking of hybridization was performed using Blocking Reagent (Sigma-Aldrich, catalogue no. 11 096 176 001). Subsequent immunofluorescence staining was performed with Anti-Digoxigenin-Fluorescein (Sigma-Aldrich, catalogue no. 11 207 741 910) in a concentration of 1:200 in 0.5% BSA/PBS for 1 h. The slides were washed twice with 1x DEPC-PBS and then mounted using Antifade Mounting Medium with DAPI (Vector, catalogue no. H-1200).
9
Binding Assay for Vip3Aa Insecticidal Protein
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Purified Vip3Aa activated protein (25 µg) was labeled with 0.5 mCi of 125I using the chloramine T method [11 (link)]. The labeled protein was separated from the excess of free 125I in a PD10 desalting column (GE Healthcare, Uppsala, Sweden) and the purity of the 125I-labeled Vip3Aa was checked by autoradiography. The specific activity of the labeled protein was 2.2 mCi/mg.
Binding assays were performed as described elsewhere [11 (link)]. Prior to being used, BBMV were centrifuged and resuspended in binding buffer (20 mM Tris, 150 mM NaCl, 1 mM MnCl2, pH 7.4, 0,04% Blocking reagent from Sigma Aldrich, St. Louis, MO, USA). Competition binding experiments were conducted by incubating 1.4 µg of BBMV protein with 0.65 nM 125I-Vip3Aa in a final volume of 0.1 mL of binding buffer for 90 min at 25 °C in the presence of increasing amounts of unlabeled Vip3Aa. After incubation, samples were centrifuged at 16,000× g for 10 min and the pellet was washed once with 500 µL of ice-cold binding buffer. Radioactivity retained in the pellet was measured in a model 2480 WIZARD2 gamma counter. Data from the competition experiments were analyzed to determine equilibrium binding parameters, dissociation constant (Kd), and concentration of binding sites (Rt) using the LIGAND software [44 (link)].
Binding assays were performed as described elsewhere [11 (link)]. Prior to being used, BBMV were centrifuged and resuspended in binding buffer (20 mM Tris, 150 mM NaCl, 1 mM MnCl2, pH 7.4, 0,04% Blocking reagent from Sigma Aldrich, St. Louis, MO, USA). Competition binding experiments were conducted by incubating 1.4 µg of BBMV protein with 0.65 nM 125I-Vip3Aa in a final volume of 0.1 mL of binding buffer for 90 min at 25 °C in the presence of increasing amounts of unlabeled Vip3Aa. After incubation, samples were centrifuged at 16,000× g for 10 min and the pellet was washed once with 500 µL of ice-cold binding buffer. Radioactivity retained in the pellet was measured in a model 2480 WIZARD2 gamma counter. Data from the competition experiments were analyzed to determine equilibrium binding parameters, dissociation constant (Kd), and concentration of binding sites (Rt) using the LIGAND software [44 (link)].
10
In Situ Hybridization of Rat Dtsp Gene
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cRNA probes for in situ hybridization of rat Dtsp were prepared as described for northern blotting analysis. Sections from PFA-fixed paraffin-embedded rat
testes were deparaffinized and hydrated. Subsequent treatment included 0.2 N HCl at room temperature for 10 min, proteinase K (diluted [1:4,000] in PBS; Takara, Shiga, Japan) at 37°C for 22
min, and 0.25% acetic anhydride in 0.1 M triethanolamine at room temperature for 10 min. Subsequently, the sections were hybridized with 1 μg/ml DIG-labeled cRNA probes overnight at 45°C. A
corresponding sense probe was used as the negative control. Inhibition of non-specific reactions involved treatment with 20 μg/ml RNaseA (Roche) at 37°C for 30 min. Sections were then
blocked with 1.5% blocking reagent (Sigma) for 1 h at room temperature, followed by two-day incubation with an alkaline-phosphatase-conjugated anti-DIG antibody (diluted 1:1,000 in 1.5%
blocking buffer, Roche) at 4°C. Finally, sections were treated with a mixture of 338 μg/ml 4-nitro blue tetrazolium chloride and 175 μg/ml 5-bromo-4-chloro-3-indolyl-phosphate (Roche)
overnight at 4°C, and signals were observed under a microscope equipped with a digital camera (DP73; Olympus, Tokyo, Japan).
testes were deparaffinized and hydrated. Subsequent treatment included 0.2 N HCl at room temperature for 10 min, proteinase K (diluted [1:4,000] in PBS; Takara, Shiga, Japan) at 37°C for 22
min, and 0.25% acetic anhydride in 0.1 M triethanolamine at room temperature for 10 min. Subsequently, the sections were hybridized with 1 μg/ml DIG-labeled cRNA probes overnight at 45°C. A
corresponding sense probe was used as the negative control. Inhibition of non-specific reactions involved treatment with 20 μg/ml RNaseA (Roche) at 37°C for 30 min. Sections were then
blocked with 1.5% blocking reagent (Sigma) for 1 h at room temperature, followed by two-day incubation with an alkaline-phosphatase-conjugated anti-DIG antibody (diluted 1:1,000 in 1.5%
blocking buffer, Roche) at 4°C. Finally, sections were treated with a mixture of 338 μg/ml 4-nitro blue tetrazolium chloride and 175 μg/ml 5-bromo-4-chloro-3-indolyl-phosphate (Roche)
overnight at 4°C, and signals were observed under a microscope equipped with a digital camera (DP73; Olympus, Tokyo, Japan).
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