Tnb blocking reagent

Manufactured by PerkinElmer

The TNB blocking reagent is a laboratory solution designed to block non-specific interactions during various analytical techniques. It is a crucial component in assays and procedures where minimizing background signals is essential for accurate results.

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Lab products found in correlation

Axiovert 200m, by Zeiss (2 mentions) Dapi vectorshield mounting medium, by Vector Laboratories (1 mentions) Gelcount colony counter, by Oxford Optronix (1 mentions) Spinning disk confocal microscope, by Zeiss (1 mentions) Triton x 100, by ICN Biomedicals (1 mentions) Fluorescence mounting medium, by Agilent Technologies (1 mentions) Colibri 7 led light source, by Zeiss (1 mentions) Axio scan z1 slide scanner, by Zeiss (1 mentions)

3 protocols using tnb blocking reagent

Quantifying Hypoxia in Cell Spheroids

Cited 1 time

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FaDu spheroids were grown using the liquid overlay technique, and HCT116 and H1299 spheroids were grown in lipidure-coated U-bottom plates (UMS Bio). Half of the medium was replaced twice per week. Spheroid hypoxia was quantified by EF5 staining, using an anti-EF5 antibody (both reagents obtained from University of Pennsylvania)7 (link). Two hundred micromolar EF5 was added to the spheroids for 6 h before fixation in 4% paraformaldehyde for 24 h, incubation in 30% sucrose for 2.5 h, and then addition of OCT (VWR, TissueTek) for cryosectioning. Spheroid sections were incubated for 40 min in TNB blocking reagent (Perkin Elmer), washed for 5 min in 1 × PBS, 0.3% Tween 20, and then incubated overnight with 70 μl of undiluted 75 μg ml⁻¹ anti-EF5 antibody. Three washes of 45 min were then performed using 1 × PBS, 0.3% Tween 20 before addition of DAPI vectorshield mounting medium (Vector Laboratories) and fluorescence microscopy (Nikon 90i, Nikon). Spheroid diameter was derived from the spheroid cross-sectional area measured by the Gelcount colony counter (Oxford Optronix) or Axiovert200M (Carl Zeiss MicroImaging GmbH).

Ashton T.M., Fokas E., Kunz-Schughart L.A., Folkes L.K., Anbalagan S., Huether M., Kelly C.J., Pirovano G., Buffa F.M., Hammond E.M., Stratford M., Muschel R.J., Higgins G.S, & McKenna W.G. (2016). The anti-malarial atovaquone increases radiosensitivity by alleviating tumour hypoxia. Nature Communications, 7, 12308.

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Immunofluorescence Assay for PKR and eif3η

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Transfected or infected cells (as specified in figure legends) seeded in 96 wells-plates were fixed with PBS containing 4% paraformaldehyde for 5 min at room temperature. Cells were then washed with PBS and permeabilized with PBS-0.1% Triton X-100 (ICN Biomedicals Inc.) for 5 min at room temperature. Cells were then blocked with TNB blocking reagent (Perkin Elmer) for 1 h at room temperature. Primary antibodies, diluted in TNB reagent, were incubated for 1 h at room temperature at the following dilutions: anti-PKR (rabbit, 18244-1-AP, Proteintech) 1:400, anti-eif3η (mouse, sc-137214 Santa Cruz) 1:800. Cells were then washed three times for 5 min in PBS-0.1% Tween 20 and incubated with species-matched secondary antibodies (Alexa Fluor 488- and 697-conjugated antibodies, Molecular Probes) 1:800 for 1 h at room temperature. Cells were washed three times for 5 min in PBS-0.2% Tween 20 and maintained in PBS-azide 0.02% until analysis. Fluorescence analysis was performed with a spinning disk confocal microscope (Zeiss, Germany). Image acquisition and processing (intensity, contrast and pseudocolours) were done with the Zen image acquisition sofware (Zeiss, Germany) using the same parameters across micrographs.

Cesaro T., Hayashi Y., Borghese F., Vertommen D., Wavreil F, & Michiels T. (2021). PKR activity modulation by phosphomimetic mutations of serine residues located three aminoacids upstream of double-stranded RNA binding motifs. Scientific Reports, 11, 9188.

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Immunofluorescence Protocol for Tissue Sections

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10 μm cryostat sections were thawed, air dried and fixed with ice-cold 100% methanol for 5 minutes. After rehydration with PBS, sections were washed twice in PBS +0.2% Tween 20 (Applied Chemicals), permeabilized with PBS +0.2% Triton X-100 (Applied Chemicals) for 3 hours and washed again with PBS +0.2% Tween 20. Blocking was performed with PBS +0.5% Tween 20 +1% TNB Blocking Reagent (Perkin Elmer), followed by incubation with primary antibody in the same buffer overnight at 4ºC. Primary antibody information and dilutions are listed in the Key Resources table. Sections were washed with PBS +0.2% Tween 20 before incubation with fluorophore-conjugated secondary antibodies at a dilution of 1:500 in PBS +0.5% Tween 20 +1% TSA Blocking Reagent +1 μg/ml DAPI at room temperature. Directly-conjugated primary antibodies were employed where indicated after an initial round of primary and secondary antibody staining, to avoid potential for cross reactivity. Finally, sections were washed with PBS +0.2% Tween and mounted with Fluorescence Mounting Medium (Dako).
Stained tissue sections were imaged with an Axio Scan.Z1 slide scanner (Zeiss) equipped with a Colibri 7 LED light source (Zeiss) using a Plan-Apochromat 20x/0.8 DIC M27 cover slip-corrected objective (Zeiss). All slides from the same staining panel were digitalized using identical acquisition settings.

Klemm F., Maas R.R., Bowman R.L., Kornete M., Soukup K., Nassiri S., Brouland J.P., Iacobuzio-Donahue C.A., Brennan C., Tabar V., Gutin P.H., Daniel R.T., Hegi M.E, & Joyce J.A. (2020). Interrogation of the Microenvironmental Landscape in Brain Tumors Reveals Disease-Specific Alterations of Immune Cells. Cell, 181(7), 1643-1660.e17.

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