Bp151 500

Manufactured by Thermo Fisher Scientific

Sourced in United States

The BP151-500 is a laboratory balance from Thermo Fisher Scientific. It is capable of measuring weights up to 500 grams with an accuracy of 0.01 grams.

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12 protocols using bp151 500

Immunofluorescence Staining Protocol

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Cells were fixed in 4% paraformaldehyde for 20mins at room temperature, rinsed 3x in PBS, permeabilized with 0.5% Triton-X100/PBS (Fisher BP151–500) for 10mins at room temperature, followed by blocked with 3% BSA/PBS (Fisher BP9706–100) for 2hrs at room temperature. Cells were subsequently treated with primary antibodies (1:200–1:400) diluted in block and incubated at 4° C overnight. Cells were rinsed 3x in PBS for 5mins at room temperature followed by secondary antibodies diluted in PBS at 1:400 for 2 hours at room temperature. Nuclei were stained with Hoechst 33342 (Invitrogen H3570) diluted in PBS and incubated for 5 minutes at room temperature. The cells were mounted with glycerol containing 1% n-propyl gallate (Sigma P3130–100G).

Han L., Choudhury S., Mich-Basso J.D., Ammanamanchi N., Ganapathy B., Suresh S., Khaladkar M., Singh J., Maehr R., Zuppo D.A., Kim J., Eberwine J.H., Wyman S.K., Wu Y, & Kühn B. (2020). Lamin B2 levels regulate polyploidization of cardiomyocyte nuclei and myocardial regeneration. Developmental cell, 53(1), 42-59.e11.

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Live-Labeling Cultured Hippocampal Neurons

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Cultured hippocampal neurons were live-labelled at DIV 21 with 50 nM scFv-Clasp either conjugated to Alexa647 (confocal imaging) or CF568 (STORM imaging) at room temperature for 20 min in aCSF (in mM: 120 NaCl, 5 KCl, 1.2 MgCl₂*6H₂O, 2 CaCl₂, 25 HEPES, 30 glucose, pH set to 7.3–7.4 with 2 M NaOH). Neurons were briefly washed 5 times over 10 min in aCSF and PBS and fixed in 4% PFA, 4% sucrose in PBS for 10 min at room temperature. Subsequently cells were washed again, permeabilised with 0.1% Triton X-100 (Fisher Bioreagents, Cat.# BP151-500) and treated with blocking solution containing 1% bovine serum albumin (Fisher Bioreagents, Cat.# BP1605-100) and 10% normal goat serum (Sigma-Aldrich, Cat.# G9023) in PBS. Neurons were incubated sequentially with primary and secondary antibody solutions prepared in PBS supplemented with 1% BSA and 10% normal goat serum for 2 h at room temperature, and washed in PBS after each incubation. For 3D STORM imaging a second fixation was performed after incubation with the secondary antibody and coverslips were stored in PBS at 4 °C until imaging. For confocal imaging, coverslips were mounted in ProLong Diamond antifade Mountant (Invitrogen, Cat.# P36961) and left to cure for 48 h at room temperature before imaging.

Watson J.F., Pinggera A., Ho H, & Greger I.H. (2021). AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP γ8 interactions. Nature Communications, 12, 5083.

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Immunofluorescent Staining Protocol for p75NGFR and Fibronectin

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The cells were fixed for 30 minutes in ice-cold 4% paraformaldehyde solution in 0.1 M
phosphate buffer (PB). The dishes were washed 4× with 0.01 M phosphate-buffered saline
(PBS), incubated in a blocking buffer consisting of 2% skimmed milk (Oxoid LP6031) and 1%
Triton X-100 (Fisher Bioreagents, BP151-500, Fairlawn, NJ, USA) in 0.01 M PBS for 60 min.
The cells were then incubated at 4°C overnight, using a cocktail of primary antibodies:
mouse monoclonal anti-nerve growth factor receptor (MAB365 p75NGFR; 1:500, Millipore,
Temecula, CA, USA) and rabbit polyclonal anti-human fibronectin (1:1000, A0245: Dako,
Denmark). Subsequently, fluorescent conjugated secondary antibodies (goat anti-mouse
Alexa-488, and goat anti-rabbit Alexa-546, both 1:500, Molecular Probes, Invitrogen) were
applied to the dishes for 1 h at room temperature. Following this, all dishes were
counterstained using Prolong Gold antifade reagent containing the nuclear dye 40,
6-diamidino-2-phenylindole (DAPI; 1mg/ml; Invitrogen, P36935).

Liadi M., Collins A., Li Y, & Li D. (2018). The Impact of Tissue Storage Conditions on Rat Olfactory Ensheathing Cell Yield and the Future Clinical Implications. Cell Transplantation, 27(9), 1320-1327.

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Immunofluorescence Labeling of Tissue Sections

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For immunofluorescence labeling, we removed the tissue sections from cryoprotectant and rinsed them in PBS before loading them into a primary antibody solution. Primary antisera (Table 3) were added to a PBS solution with 0.25% Triton X‐100 (BP151‐500, Fisher), 2% normal donkey serum (NDS, 017‐000‐121, Jackson ImmunoResearch), and 0.05% sodium azide (14314, Alfa Aesar) as a preservative (PBT‐NDS‐azide). We incubated these sections overnight at room temperature on a tissue shaker. The following morning, the sections were washed 3× in PBS and incubated for 2 h at room temperature in PBT‐NDS‐azide solution containing species‐specific donkey secondary antibodies conjugated to Cy3, Cy5, or Alexa Fluor 488, or biotin (diluted 1:500−1000; Jackson ImmunoResearch) in PBT‐NDS‐azide. If a biotinylated secondary antibody was used, tissue sections were then washed three times and incubated for 2 h in streptavidin‐Cy5 (#SA1011, Invitrogen) diluted 1:1000 in PBT‐NDS‐azide. The sections were then washed three times in PBS, mounted on glass slides (#2575‐PLUS; Brain Research Laboratories), dried, and then coverslipped using Vectashield (Vector Laboratories). Slides were stored at 4°C until imaging.

Huang D., Zhang R., Gasparini S., McDonough M.C., Paradee W.J, & Geerling J.C. (2022). Neuropeptide S (NPS) neurons: Parabrachial identity and novel distributions. The Journal of Comparative Neurology, 530(18), 3157-3178.

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Immunofluorescence Staining of Transfected HMC3 Cells

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Transfected HMC3 cells were fixed with 10% neutral buffered formalin (Fisher Scientific SF100–4) for 30 minutes then blocked and permeabilized for 30 minutes with 10% goat serum (Sigma S26-LITER), 0.1% Triton X-100 (Fisher Scientific BP151–500) in PBS (Fisher BioReagents BP665–1). Primary and secondary antibodies were diluted in the same blocking and permeabilization buffer and incubated at room temperature for 90 minutes. Cells were washed three times in blocking and permeabilization buffer between primary and secondary antibodies, and three times in PBS prior to coverslip mounting with Prolong Glass with NucBlue mounting media (Invitrogen P36981) and high-tolerance No. 1.5 coverglass (ThorLabs CG15KH1). Images were acquired using a Nikon A1R HD inverted confocal microscope with a 60X oil objective and NIS Elements AR software.

Shaw B.C., Snider H.C., Turner A.K., Zajac D.J., Simpson J.F, & Estus S. (2022). An alternatively spliced TREM2 isoform lacking the ligand binding domain is expressed in human brain. Journal of Alzheimer's disease : JAD, 87(4), 1647-1657.

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Immunophenotyping and Cell Function Analysis

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Cells were resuspended in a master mix made of PBS, viability dye (Tonbo, 13–0865-T500), and surface stain antibodies, then incubated at RT in the dark for 20 minutes. The cells were washed and incubated in 2% formaldehyde (Thermo, 28908) at 37 °C for 10 minutes. After incubation, the cells were washed and resuspended in 0.04% Triton (Fisher, BP151–500) at RT in the dark for exactly 7 minutes. After 7 minutes, the cells were washed in PBS + 2% FBS (PEAK Serum, PS-FB1) + 2 mM EDTA (Fisher, BP2482–1) + 2% BSA (MP Biomedicals, 810681) (FACS buffer) and stained with an internal master mix in FACS buffer. Depending on the antibodies used, the cells were then incubated at RT in the dark for 30–120 minutes. After incubation, the cells were washed with FACS buffer, washed again with PBS, and resuspended in PBS for flow cytometry. Samples were run on a CytoFLEX flow cytometer (Beckman-Coulter) and analyzed via FlowJo 10.8.0 (FlowJo) to determine the extent of gene ablation and the effect of gene ablation on cell function.

Dahlvang J.D., Dick J.K., Sangala J.A., Kennedy P.R., Pomeroy E.J., Snyder K.M., Moushon J.M., Thefaine C.E., Wu J., Hamilton S.E., Felices M., Miller J.S., Walcheck B., Webber B.R., Moriarity B.S, & Hart G.T. (2023). Ablation of SYK kinase from expanded primary human Natural Killer cells via CRISPR/Cas9 enhances cytotoxicity and cytokine production. Journal of immunology (Baltimore, Md. : 1950), 210(8), 1108-1122.

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Immunofluorescence Labeling Protocol

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For immunofluorescence labeling, we removed the tissue sections from cryoprotectant and rinsed them in PBS before loading them into a primary antibody solution. Primary antisera (Table 3) were added to a PBS solution with 0.25% Triton X-100 (BP151–500, Fisher), 2% normal donkey serum (NDS, 017-000-121, Jackson ImmunoResearch), and 0.05% sodium azide (14314, Alfa Aesar) as a preservative (PBT-NDS-azide). We incubated these sections overnight at room temperature on a tissue shaker. The following morning, the sections were washed 3× in PBS and incubated for 2 hours at room temperature in PBT-NDS-azide solution containing species-specific donkey secondary antibodies conjugated to Cy3, Cy5, or Alexa Fluor 488, or biotin (diluted 1:500−1000; Jackson ImmunoResearch) in PBT-NDS-azide. If a biotinylated secondary antibody was used, tissue sections were then washed three times and incubated for two hours in streptavidin-Cy5 (#SA1011, Invitrogen) diluted 1:1,000 in PBT-NDS-azide. The sections were then washed three times in PBS, mounted on glass slides (#2575-PLUS; Brain Research Laboratories), dried, and then coverslipped using Vectashield (Vector Laboratories). Slides were stored at 4 °C until imaging.

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Immunostaining of Retinal Neurons

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After the dissection, the retina was washed 3 times with PBS at room temperature for 5 min each round. Then the retina was incubated with a blocking solution (5% horse serum (H1138, Sigma Aldrich) + 0.3% Triton X-100 (BP151–500, Fisher Scientific) in PBS) at room temperature for 1 h and then incubated with primary antibodies goat anti-VAChT (1:800, Millipore, ABN100), goat anti-ChAT (1:200, AB144P, Millipore), rabbit anti-RFP (1:1000, ab62341, Abcam) at 4 °C for 5 days. Next, the retina was washed 3 times with PBS at room temperature for 30 min each round and then incubated with secondary antibodies Donkey anti-goat-Alexa Fluor 488 (1:500, A32814, Invitrogen) and Donkey anti-rabbit-Alexa Fluor 594 (1:1000, A32754, Invitrogen) at room temperature for 2 h. Then the retina was washed 3 times with PBS at room temperature for 5 min each round and mounted in a homemade mounting medium (80% glycerol (BDH1172, VWR), 1% DABCO (D27802, Sigma Aldrich) in PBS, pH 8.6). The above commercial antibodies have been previously validated by their manufactures and were well characterized in the literature.

Liu J., He Y., Lavoie A., Bouvier G, & Liu B.H. (2023). A direction-selective cortico-brainstem pathway adaptively modulates innate behaviors. Nature Communications, 14, 8467.

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Immunofluorescence analysis of AMPK and SAPS3

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Cells were washed twice with PBS and fixed with 4% formaldehyde (Thermo Scientific, 28908) in PBS for 15 min at room temperature. Cells were washed again three times with PBS and permeabilized with 0.1% TritonX-100 (Fisher, BP151-500) in PBS at room temperature for 10 min. Then cells were washed three times with PBS and blocked with 3% BSA in PBS for 1 h. Thereafter, cells were incubated with anti-AMPKα (Thermo Scientific, MA537501, 1:50) and anti-SAPS3 (Thermo Scientific, 16944-1-AP, 1:100) antibodies overnight at 4 °C. Cells were washed three times with PBS and incubated with goat anti-rabbit 594 (Invitrogen, A11037, 1:500) and goat anti-mouse 488 (Invitrogen, A11029, 1:500) antibodies for 1 h at room temperature. After that, cells were washed three times and mounted with the Prolong Gold antifade reagent with DAPI (Invitrogen, P36931). The slides were visualized with Zeiss LSM 900 Airyscan.

Yang Y., Reid M.A., Hanse E.A., Li H., Li Y., Ruiz B.I., Fan Q, & Kong M. (2023). SAPS3 subunit of protein phosphatase 6 is an AMPK inhibitor and controls metabolic homeostasis upon dietary challenge in male mice. Nature Communications, 14, 1368.

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Immunofluorescence Staining of Transfected HMC3 Cells

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Transfected HMC3 cells were fixed with 10% neutral buffered formalin (Fisher Scientific SF100-4) for 30 minutes then blocked and permeabilized for 30 minutes with 10% goat serum (Sigma S26-LITER), 0.1% Triton X-100 (Fisher Scientific BP151-500) in PBS (Fisher BioReagents BP665-1). Primary and secondary antibodies were diluted in the same blocking and permeabilization buffer and incubated at room temperature for 90 minutes. Cells were washed three times in blocking and permeabilization buffer between primary and secondary antibodies, and three times in PBS prior to coverslip mounting with Prolong Glass with NucBlue mounting media (Invitrogen P36981) and high-tolerance No. 1.5 coverglass (ThorLabs CG15KH1). Images were acquired using a Nikon A1R HD inverted confocal microscope with a 60X oil objective and NIS Elements AR software.

Shaw B.C., Snider H.C., Turner A., Zajac D.J., Simpson J.F., & Estus S. (2021). An alternatively spliced TREM2 isoform lacking the ligand binding domain is expressed in human brain.

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