Buffer w

Manufactured by NanoString

Buffer W is a laboratory reagent used to maintain and stabilize the pH and ionic conditions of biological samples. It is a key component in various biomolecular analysis and purification protocols.

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Lab products found in correlation

Syto13, by NanoString (1 mentions) Tbs t, by Thermo Fisher Scientific (1 mentions) Tween 20 tbst, by Cell Signaling Technology (1 mentions) Formaldehyde, by Thermo Fisher Scientific (1 mentions) Geomx digital spatial profiler, by NanoString (1 mentions) Citrisolv, by Decon Labs (1 mentions) Ab196147, by Novus Biologicals (1 mentions) Mabn92 af647, by Abcam (1 mentions) Nbp2 33184af532, by Thermo Fisher Scientific (1 mentions) Bond platform, by Leica (1 mentions) S7575, by Thermo Fisher Scientific (1 mentions) Syto83, by Thermo Fisher Scientific (1 mentions)

3 protocols using buffer w

GeoMx DSP Tissue Immunostaining Protocol

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FFPE tissue sections (5 µm thick) were processed according to the GeoMx® Digital Spatial Profiler (DSP) protocol (NanoString Technologies, Inc.). Briefly, tissue was deparaffinized and rehydrated with CitriSolv (Decon Labs), ethanol and ddH₂O washes at room temperature. Antigen retrieval was performed in citrate buffer pH 6.0 (Millipore) at ~ 115 °C under high pressure and washed in tris-buffered saline with Tween 20 (TBS-T) (Cell signaling Technology). Tissue was blocked with Buffer W (Nanostring) for 1 h at room temperature. Slides were incubated at 4 °C overnight with antibody mixture including fluorescently tagged antibodies for IBA1-AF532 (Nanostring Technologies, Inc. #121,300,306) and CD45-AF594 (NanoString Technologies, Inc. #121,300,301) and the following oligo-tagged NanoString GeoMx human detection antibody mixtures: Immune Core Profiling, Immune Cell Typing, Immune Activation, and Cell Death (see Supplemental Material for list of detection targets). Slides were washed with TBS-T, fixed with 4% formaldehyde (Invitrogen) for 1 h at room temperature, then re-washed with TBS-T. Nuclei were stained with SYTO 13 (NanoString).

Westfall J.J., Schwind W.N., Sran S., Navarro J.B., Leonard J., Pindrik J.A., Pierson C.R., Boué D.R., Koboldt D.C., Ostendorf A.P., Wilson R.K., Mardis E.R., Miller K.E, & Bedrosian T.A. (2022). Molecular and spatial heterogeneity of microglia in Rasmussen encephalitis. Acta Neuropathologica Communications, 10, 168.

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GeoMx DSP Slide Preparation Protocol

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Slides were prepared by baking in a drying oven and then processed using the Leica BOND platform (Leica Biosystems) as specified by the NanoString GeoMx DSP Slide Preparation User Manual. Briefly, slides were deparaffinized with xylene then rehydrated through a graded ethanol series. Targets were exposed by incubating the slides in a pressure cooker at 100°C in 1X Tris-EDTA buffer, pH9 followed by proteinase K digestion. Next, the GeoMx NGS-RNA Probe Mix (NanoString Technologies) was applied to each slide, and slides were incubated at 37°C for 16-24 hours. This probe mix contained barcoded oligonucleotide probes against 1,700 gene targets and included internal positive and negative control probes. After washing, the slides were then blocked in Buffer W (NanoString Technologies) and stained with morphology markers for 1 hour. The fluorescently conjugated morphology markers used are as follows: Iba1 at 1:75 dilution (EMD Millipore, MABN92-AF647), CD3 at 1:50 dilution (Abcam, ab196147), GFAP at 1:3000 dilution (Novus Biologicals, NBP2-33184AF532), and SYTO 13 nuclei acid stain at 1:5 dilution (Thermo Scientific S7575).

Kim Y., Danaher P., Cimino P.J., Hurth K., Warren S., Glod J., Beechem J.M., Zada G, & McEachron T.A. (2023). Highly multiplexed spatially resolved proteomic and transcriptional profiling of the glioblastoma microenvironment using archived formalin-fixed paraffin-embedded specimens.. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 36(1), 100034.

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Spatial Transcriptomic Analysis of Tissue

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Tissue sections were then incubated overnight at 37°C with Mouse Whole Transcriptome Atlas RNA detection probes in Buffer R (NanoString Technologies) using a Hyb EZ II hybridization oven (ACD). The DNA oligonucleotide probes were composed of a sequence complementary to a target mRNA joined to a sequencing oligonucleotide via an ultraviolet photocleavable linker. Each unique sequencing oligonucleotide consisted of a gene-specific barcode, Unique Molecular Index (UMI) and primer binding sites. During incubation, slides were covered with HybriSlip Hybridization Covers (Grace BioLabs, 714022). Following incubation, HybriSlip covers were gently removed, and slides were washed in 50% formamide and 2X saline sodium citrate (SSC) for 25 min at 37°C, twice. Tissues were then washed for 5 min in 2X SSC, subsequently blocked in Buffer W (Nanostring Technologies) for 30 min at room temperature in a humidity chamber, and then incubated in 500nM Syto83 (ThermoFisher) for visualization of nuclei. Next, slides were washed twice in fresh 2X SSC then loaded on the GeoMx Digital Spatial Profiler.

Roman K.M., Dinasarapu A.R., VanSchoiack A., Ross P.M., Kroeppler D., Jinnah H.A, & Hess E.J. (2023). Spiny projection neurons exhibit transcriptional signatures within subregions of the dorsal striatum. Cell reports, 42(11), 113435.

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