All slides were post-fixed in 4% PFA for 10 min, washed in PBT (0.1% Tween-20 in 1× PBS), permeabilized in 0.5% proteinase K, and acetylated in 0.1 M TEA (1.86% triethanolamine, 0.4% 10N NaOH, 0.5% acetic anhydride). Slides were incubated with probe in hybridization solution (50% formamide, 5× SSC, 2% blocking reagent (Roche, Mannheim, Germany; 11096176001), 0.1% Triton, 0.15 CHAPS, 1mg/ml tRNA from yeast, 5mM EDTA, 50ug/ml heparin) overnight at 65°C in a box humidified with a solution of 50% formamide and 5× SSC. Following an incubation in 1× SSC/50% formamide for 30 minutes at 65°C, slides were treated with RNaseA, and placed through a series of washes with SSC and MABT (100mM maleic acid, 150mM NaCl, 0.1% Tween-20, pH 7.5). After blocking for 1 hour at room temperature in 2% blocking reagent (Roche, Indianapolis IN) and 20% goat serum, slides were incubated overnight at 4°C with anti-DIG antibody (1:2500; Roche #11093274910, Indianapolis IN) in 2% blocking reagent and 5% goat serum. Slides were washed with five 1 hour washes in MABT and one 10min wash in NTM (100mM Tris pH 9.5, 100mM NaCl, 50mM MgCl2) before developing in the dark overnight with BM Purple (Roche, 11442074001, Indianapolis IN) at room temperature. Finally, slides were rinsed with NTM, washed 3× in 1× PBS, fixed in PFA for 30min, dehydrated, and mounted with Permount.
Blocking reagent
Manufactured by Roche
Sourced in Germany, United States, Switzerland, United Kingdom
Blocking reagent is a laboratory product used to prevent non-specific binding in various immunoassay techniques. It helps reduce background signals and improve the specificity of target analyte detection.
Automatically generated - may contain errors
Lab products found in correlation
Nbt bcip, by Roche (14 mentions)
Axio observer z1, by Zeiss (12 mentions)
Lsm 700, by Zeiss (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions)
Dig rna labeling kit, by Roche (7 mentions)
Nbt bcip solution, by Roche (7 mentions)
Anti dig antibody, by Roche (7 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Proteinase k, by Roche (6 mentions)
Cdp star, by Roche (6 mentions)
Bm purple, by Roche (6 mentions)
Alkaline phosphatase conjugated anti dig antibody, by Roche (6 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (6 mentions)
Vectashield, by Vector Laboratories (5 mentions)
Anti dig ap antibody, by Roche (5 mentions)
Positively charged nylon membrane, by Roche (5 mentions)
Anti dig alkaline phosphatase antibody, by Roche (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Formamide, by Merck Group (4 mentions)
Proteinase k, by Merck Group (4 mentions)
280 protocols using blocking reagent
1
In-situ Hybridization Protocol for Tissue Sections
2
Osteomodulin Expression in Osteoarthritis Serum
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Serum of 12 men were used in western-blotting experiments, 6 healthy (mean age 43, range 22–61) and 6 suffering of severe OA just before TKR (mean age 67, range 44–82). Before experiment serum were depleted in IgG and albumin using ProteoPrep kits (Sigma). During this process serum is diluted by approximately 3.3-fold. Supernatants of three independent Subchondral osteoblast culture were 20-fold concentrated using Amicon Ultra 3kDa 2 ml column (Millipore). Recombinant human OMD (R&D systems, 2884-AD) was used as positive control. Depleted serum (15μl) or osteoblasts concentrated supernatant (6 μl = 6 μg total proteins) were fractioned by electrophoresis on a polyacrylamide gel (9%) and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked overnight 4°C with Roche Blocking Reagent (Villevorde, Belgium). Membranes were then incubated overnight at 4°C with a biotinylated polyclonal goat antiserum, affinity purified, raised against the whole osteomodulin protein (BAF2884, R&D systems, Minneapolis, USA) 1:200 dilution in 0.5% Roche Blocking Reagent. Streptavidin-Horse-radish peroxidase (HRP) (1:2500 dilution) was used as detection (Roche). The reaction was revealed with Luminata classico Western blotting substrate (Millipore) and capture with an ImageQuantLAS4000 (Amersham).
3
Immunofluorescent Labeling of C17 and Laminin-332
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Cryosections of 8 µm were fixed with acetone:methanol (1:1) at −20 °C for 15 min and washed twice with PBS for 5 min. The slides were then incubated for 1.5 h with a human-specific anti-C17 antibody (#184996; Abcam, Cambridge, United Kingdom) diluted 1:500 in 1× blocking reagent (Roche Diagnostics GmbH, Mannheim, Germany) in TBS-T. After three washing steps with PBS, secondary antibody Alexa Fluor 549 goat anti-rabbit IgG (H + L) (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:500 in PBS and DAPI (4′,6-Diamidino-2-phenylindol) (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:1000 in PBS for 1 h at room temperature. After three washing steps with PBS for 5 min, cryosections were covered with a DAKO fluorescent mounting medium (Agilent, Santa Clara, CA, USA). For laminin-332 staining, slides were incubated for 1.5 h with a human-specific anti-laminin alpha 3/laminin-5 antibody (#MAB2144; R&D systems, Minneapolis, MN, USA) diluted 1:2000 in 1× blocking reagent (Roche Diagnostics GmbH, Mannheim, Germany) in TBS-TX. Staining with secondary antibody and DAPI was performed in the same way as for C17. Cryosections were analyzed using the confocal laser scanning microscope Axio Observer Z1 attached to LSM700 (Carl Zeiss).
4
Immunofluorescence Staining of Planarian Neurons
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The same procedure as that used for WISH was used for sample preparation. Fixed pharynxes were blocked with 1% Blocking reagent (Roche) in PBST for 30 min at 4°C and then incubated with mouse anti-planarian synaptotagmin (SYT) (1:1000 dilution) (19 (link)) and rabbit anti-planarian cytochrome b561 (CYT b561) (1:1000 dilution) (34 (link)) antibody overnight at 4°C. The samples were washed with PBST for 30 min four times, and signals were detected with Alexa Fluor 488– or Alexa Fluor 594–conjugated goat anti-mouse or anti-rabbit immunoglobulin G (IgG) (1:1000 dilution) (Invitrogen) in 1% Blocking reagent (Roche) in PBST overnight at 4°C in the dark. The samples were also incubated with Hoechst 33342 (10 μg/ml; Thermo Fisher Scientific) at the same time.
5
Probe Sequence and Fluorochrome Effects on FISH Specificity
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To determine if the sequence of the oligonucleotide probe contributes to non-specific binding, three probes were assessed: EUB338 mix probes, NonEUB338 and a Vib-GV (5′–AGG CCA CAA CCT CCA AGT AG-3′; Giuliano et al., 1999 (link)). To determine if the fluorochrome attached to the oligonucleotide probe affects non-specific binding, three flurochromes were assessed: Atto 647 (excitation λ = 645, emission λ = 669) Cy3 (excitation λ = 548, emission λ = 561) and Cy5 (excitation λ = 647, emission λ = 665) (i.e., Cy3-labeled EUB338 mix, Cy3-labeled NonEUB338, Cy5-labeled NonEUB338 and Atto 647-labelled Vib-GV). To assess the utility of incorporating hybridising agents in the FISH workflow to avoid non-specific binding, “Blocking Reagent” (Roche, Germany) was added to the hybridisation buffer (30% formamide, 0.9M NaCl, 20 mM Tris–HCl with adjusted pH8.0, 0.01% SDS, 10% “Blocking Reagent” with maleic acid buffer), alongside Cy3-labelled NonEUB338 probe.
6
Visualizing H+-ATPase in Zebrafish
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The number of the HR cells was counted in the yolk-sac membrane of the control larvae and pck1 morphants injected with water or glucose into the yolk. At 3 dpf, the larvae were treated with acidic freshwater for 6 h, fixed in 4% paraformaldehyde (PFA) in PBS, and kept in 70% ethanol until subsequent procedure was performed. The samples were washed in PBST, blocked in the blocking reagent (Roche Applied Science), and incubated overnight in the anti-zebrafish H+-ATPase antibody diluted 1:1000 in the blocking reagent. After brief washing, the samples were further incubated in goat anti-rabbit IgG antibody labeled with Alexa 488 (Life Technologies) diluted 1:1000 in PBS. The fluorescent images of the larvae were obtained by a Leica TCS-SP5 confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany). In the fluorescent micrographs, the number of HR cells was counted in the area corresponding to 15,000 μm2 at the yolk-sac membrane.
7
Western Blot Analysis Protocol
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Western blot analysis was performed as previously described24 (link). Samples from cell lysates or tissue lysates were resolved by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membrane. After 1 h blocking at room temperature using 10% blocking reagent (Roche), membrane was incubated overnight with primary antibody in Tris-buffered saline solution/Tween (TBST) containing 10% blocking reagent at 4 °C. After the incubation, membrane was washed three times in TBST and incubated with secondary antibody for 1 h at room temperature. After three-time washing in TBST, membrane was developed using a chemiluminescence assay system (Roche) and exposed to Kodak exposure films. Relative protein levels were quantified by Image J program. For stripping, membrane was vigorously shaken in stripping buffer (62.5 mM Tris-HCl, pH 6.7; 2% SDS; 100 mM 2-mecaptomethanol) at 50 °C for 20 min. After stripping, membrane was washed three times in TBST and re-blotted with other antibodies with the procedures as described above.
8
Western Blot Analysis Protocol
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Western blot analysis was performed as previously described (Sun et al. 2014 (link)). Samples from cell lysates or tissue lysates were resolved by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membrane. After 1 h blocking at room temperature using 10% blocking reagent (Roche), membrane was incubated overnight with primary antibody in Tris-buffered saline solution/Tween (TBST) containing 10% blocking reagent at 4 °C. After the incubation, membrane was washed three times in TBST and incubated with secondary antibody for 1 h at room temperature. After three-time washing in TBST, membrane was developed using a chemiluminescence assay system (Roche) and exposed to Kodak exposure films. Relative protein levels were quantified by Image J program. For stripping, membrane was vigorously shaken in stripping buffer (62.5 mM Tris-HCl, pH 6.7; 2% SDS; 100 mM 2-mecaptomethanol) at 50 °C for 20 min. After stripping, membrane was washed three times in TBST.
9
Western Blot Protein Extraction and Analysis
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Cells were lysed using RIPA lysis buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, supplemented with protease inhibitor cocktail; Sigma) by pipetting 50 times and rapid vortexing. The lysates were cleared by centrifuging for 15 min at 16,100g at 4 °C. Total protein was determined using the bicinchoninic acid (BCA) method (Micro BCA Protein assay kit, Thermo Fisher Scientific; MA, USA). Fifty micrograms of lysates were loaded on tris-glycine gels and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis then transferred onto Hybond PVDF 0.22 µm membrane (Millenium Science; Victoria, Australia). Membranes were blocked with 1% blocking reagent (Roche) in phosphate-buffered saline (PBS), and probed with primary antibodies then horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in 1% blocking reagent (Roche) in PBS with 0.1% Tween-20 (Sigma). SuperSignal West Dura extended duration substrate (Thermo Fisher Scientific) was used for detection.
10
Microarray Hybridization and Signal Detection
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Hybridization of amplicons to probes on microarrays was performed according to a previously described protocol (Hsiao et al., 2005 ), with modifications. Briefly, 4 μl of labelled amplicons were added to 220 μl of hybridization solution [5 × SSC (v/v), 2% blocking reagent (w/v; Roche Applied Science), 0.1% N‐lauroylsarcosine (w/v) and 0.02% SDS (w/v)], denatured with boiling water for 7 min, and immediately chilled on ice for 10 min. Hybridization was conducted at 58°C for 40 min, and arrays were then washed twice at 56°C for 5 min each with 0.25 × SSC to remove non‐hybridized PCR products, and incubated for 30 min with 200 μl of blocking solution [1% (wt/vol) blocking reagent dissolved in maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5)] containing either anti‐DIG‐AP (1:5,000 dilution; Roche Applied Science) or streptavidin‐AP (1:1,000 dilution; Roche Applied Science). Signals were colour developed with 500 μl nitroblue tetrazolium (NBT)/5‐bromo‐6‐chloro‐3‐indolyl phosphate, p‐toluidine salt (BCIP) solution (Roche Applied Science) at room temperature without shaking. PVC chip signals were captured and analysed with Dr AiM reader (Dr Chip Biotechnology), while nylon membrane signals were captured with a BioSpectrum Imaging System (UVP, Upland, CA, USA) and analysed with Vision Works LS Analysis Software v6.5.2 (UVP).
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