sm-FISH probe pools were generated to detect seven RNA + segments of the SARS-CoV-2 genome (NCBI reference: NC_045512.2). Each probe pool used (~ 48 oligo probes per gene, each probe 20 nucleotides in length) is listed in Additional file 1 : Table S1. Probes were designed using Stellaris™ probe designer (Biosearch Technologies LGC, Petaluma, CA) and then synthesized and purchased from Biosearch Technologies (the probe sequences can be provided upon request). The 3ʹ-end of each probe was modified with an amine group and coupled to Texas Red-X (Fisher Scientific). Coupled probes were ethanol precipitated and purified on an HPLC column to isolate oligonucleotides linked to the fluorophore, as described previously [38 (link)]. The seven sets of probes were diluted at 25 ng/ul, pooled each set with equal volumes together, and used at 25 ng per hybridization reaction (50 ul).
Texas red x
Manufactured by Thermo Fisher Scientific
Sourced in United States
Texas Red-X is a fluorescent dye commonly used in biological research applications. It is designed to emit a red-orange fluorescence when excited by light at appropriate wavelengths. The dye can be used for various labeling and detection purposes in techniques such as flow cytometry, immunohistochemistry, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
Stellaris probe designer, by Biosearch Technologies (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Quasar 670, by Biosearch Technologies (2 mentions)
Chroma tide alexa fluor 488 5 dutp, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Depsipher, by R&D Systems (1 mentions)
Anti lamp1 primary antibody, by Abcam (1 mentions)
Mouse anti gfp, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Dapi nuclear stain, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Triton x 100 solution, by Merck Group (1 mentions)
Anti ki67, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Ab24758, by Abcam (1 mentions)
Ab8898, by Abcam (1 mentions)
27 protocols using texas red x
1
SARS-CoV-2 RNA Detection by sm-FISH
2
Detecting SARS-CoV-2 RNA Transcripts Using smFISH
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smFISH probe pools were generated to detect seven different RNA+ segments of the SARS-CoV-2 genome (Accession number: NC_045512.2). Each probe pool used (48 probes per pool, each probe 20 nucleotides in length) is listed in Supplementary Table S2 . Probes were designed using Stellaris™ probe designer (Biosearch Technologies LGC, Petaluma, CA, USA) and then synthesized and purchased from Biosearch Technologies (the probe sequences are available upon request). The 3′-end of each probe was modified with an amine group and coupled to Texas Red-X (Fisher Scientific, Hampton, NH, USA). Coupled probes were ethanol precipitated and purified on an HPLC column to isolate oligonucleotides linked to the fluorophore, as described previously [55 (link)]. The seven sets of probes were diluted to 25 ng/µL, pooled together, and used at 25 ng per hybridization reaction (50 µL). The organization of the SARS-CoV-2 genome and the target region of the smFISH probe are shown in Supplementary Figure S1 .
3
SARS-CoV-2 Transcriptome Profiling by sm-FISH
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sm-FISH probe pools were generated to detect seven RNA + segments of the SARS-CoV-2 genome (NCBI reference: NC_045512.2). Each probe pool used (~48 oligo probes per gene, each probe 20 nucleotides in length) is listed in TABLE S1 . Probes were designed using Stellaris™ probe designer (Biosearch Technologies LGC, Petaluma, CA) and then synthesized and purchased from Biosearch Technologies (the probe sequences can be provided upon request). The 3’-end of each probe was modified with an amine group and coupled to Texas Red-X (Fisher Scientific). Coupled probes were ethanol precipitated and purified on an HPLC column to isolate oligonucleotides linked to the fluorophore, as described previously [38 (link)]. The seven sets of probes were diluted at 25 ng/ul, pooled each set with equal volumes together, and used at 25 ng per hybridization reaction (50 ul).
4
Apoptosis Detection in Rat Cardiomyocytes
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Ventricular myocytes from 1 to 3 day-old Sprague-Dawley rats were enzymatically dissociated, purified by Percoll gradient centrifugation and pre-plating, and cultured as described (Oh et al., 2003 (link)). To detect hypodiploid DNA, flow cytometry was performed (XL or LSRII, BD Biosciences), using propidium iodide to measure DNA content and sampling > 5000 cells for each histogram. Myocyte identity was confirmed using FITC-conjugated MF-20 antibody to sarcomeric MyHC (R&D Systems). To detect dissipation of mitochondrial membrane potential (ΔΨm), cells were incubated for 60 min in 5 μg ml-1 5, 5′, 6, 6’-tetrachloro-1, 1’, 3, 3′-tetraethylbenzimidazolyl carbocyanine iodide (DePsipher; R&D Systems). When ΔΨm is intact, mitochondrial uptake and aggregation of the dye result in red fluorescence; when ΔΨm is disturbed, the dye diffuses to the cytoplasm and reverts to its monomeric green form. Myocyte identity was confirmed using mouse antibody to sarcomeric tropomyosin (Sigma-Aldrich) conjugated with Texas Red-X (Molecular Probes).
5
Immunofluorescent Staining of Neuron-Glia Cultures
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At 72 h post-transfection, neuron-glia mixed cultures were fixed for 15 min in a 4 % paraformaldehyde solution at room temperature. After fixation, the cells were washed once with phosphate-buffered saline (PBS), and non-specific protein binding sites were blocked with 2 % bovine serum albumin (Sigma) for 45 min at 37 °C. The following primary antibodies were used: mouse anti-GFP (final dilution, 1:200; Molecular Probes), anti-LAMP1 primary antibody (final dilution of 1:200; Abcam), isolectin GS-IB4 from Griffonia simplicifolia, Alexa Fluor® 594 conjugate (final dilution of 1:100; Molecular Probes), anti-IL-1β (MAB501; final dilution, 1:100; Abcam), and OX-6 (anti-MHC II) (final dilution, 1:100; BD Pharmingen). Primary antibodies were made up in PBS, with 1 % Triton X-100 for permeabilization, and were incubated overnight at 4 °C. After three 5-min washes in PBS, the sections were incubated with the relevant secondary antibodies: Alexa Fluor® 488 (final dilution, 1:200; Molecular Probes) or Texas Red® X (final dilution, 1:200; Molecular Probes). All secondary antibodies were incubated overnight at 4 °C. After three 5-min washes in PBS, the samples were mounted with DAKO Fluorescent Mounting Medium. No staining was detected in the absence of the primary or secondary antibodies. Some preparations were counter-labeled with DAPI nuclear stain (5 μM; Molecular Probes).
6
Immunostaining of HUVEC cells after γ-irradiation
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HUVEC were immunostained after exposure to sham, 0.5 Gy, 1 Gy and 2 Gy of 60Co irradiation. Briefly, 30 min after irradiation, cells were fixed with 4% paraformaldehyde in PBS for 15 min, washed with PBS (Life Technologies), permeabilized in 0.5% Triton X-100 solution (Sigma Aldrich) for 5 min, and then washed 3 more times with PBS. The cells were then incubated for 1 h with primary antibodies, either a mix of mouse IgG1 monoclonal anti-phospho-histone H2AX (Ser139) antibody (clone JBW301, Upstate) and/or rabbit polyclonal anti-KI67 (Life Technologies). Cells were then washed 3 more times and incubated for 1 h with a secondary antibody, i.e., a mix of goat anti-mouse IgG1 (γ1) coupled to Alexa Fluor® 488 (2 mg.mL-1, Life Technologies) and/or goat anti-rabbit IgG (H+L) coupled to Texas Red®-X (2 mg.mL-1, Life Technologies). Cells were washed as described above before applying ProLong® Gold Antifade mountant with DAPI (Life Technologies).
7
Immunofluorescence Staining of Cellular Markers
8
Calcium-Independent Desmosome Assay
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Primary antibodies and dilutions used were: anti-desmoplakin I and II (clone 11-5F, D. R. Garrod), 1:400; anti-plakoglobin (clone 15F11, Sigma), 1:100; antidesmocollin 2 and 3 (clone 7G6, Thermofisher), 1:100; anti-keratin 8 (clone LE41, E. B. Lane), used as hybridoma culture supernatant; anti-plakophilin 1 (PP1-5C2, Abnova), 1:50. Secondary antibodies conjugated to Alexa Fluor 488, 594 or 647 were all from Thermofisher (used at 1:500). Alexa Fluor 488-(1:500), Texas-Red-X-(1:500) and Alexa Fluor 647-conjugated (1:200) phalloidin were from Life Technologies.
Calcium switch assay MDCK cells were cultured in SM. They were washed thrice with calcium-and magnesium-free PBS (Gibco) and incubated with calcium-free DMEM (Gibco) supplemented with 10% chelated FBS and 3 mM EGTA (Sigma-Aldrich), herein referred to as "+EGTA" for 1 h (unless specified otherwise) at 37°C 5% humidified CO2. Calcium sensitivity of desmosomes was quantified as previously described (Wallis et al., 2000) . In brief, following Ca 2+ -chelation, the cells were fixed in ice-cold methanol for 10 min and stained for desmoplakin by immunofluorescence. Cells which remained attached by desmoplakin positive projections were scored as having calcium-independent desmosomes and expressed proportional to the total cell number.
Calcium switch assay MDCK cells were cultured in SM. They were washed thrice with calcium-and magnesium-free PBS (Gibco) and incubated with calcium-free DMEM (Gibco) supplemented with 10% chelated FBS and 3 mM EGTA (Sigma-Aldrich), herein referred to as "+EGTA" for 1 h (unless specified otherwise) at 37°C 5% humidified CO2. Calcium sensitivity of desmosomes was quantified as previously described (Wallis et al., 2000) . In brief, following Ca 2+ -chelation, the cells were fixed in ice-cold methanol for 10 min and stained for desmoplakin by immunofluorescence. Cells which remained attached by desmoplakin positive projections were scored as having calcium-independent desmosomes and expressed proportional to the total cell number.
9
Synchronized Phagocytosis Assay with Labeled POS
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POS were purified from freshly obtained porcine eyes from a local slaughterhouse according to established procedures [31 (link)]. Fluorescent dye labeling was performed immediately before use as described with 0.1 mg/mL FITC (FITC Isomer I, #F1906, Thermofisher), or 0.01 mg/mL Texas Red-X (mixed isomers, #T6134, Thermofisher) [31 (link)]. Synchronized phagocytosis assays were performed as described in detail previously [32 (link)]. In brief, cells on glass coverslips in multi-well plates were fed POS at a density of 10 particles/cell in DMEM supplemented with 1.25 μg/mL recombinant mouse MFG-E8 (#2805-MF-50/CF, R & D Systems, Minneapolis, MN, USA) for the duration of experiments at 37 °C, or for 1 h at 20 °C to allow only POS binding in synchronized phagocytosis assays [33 (link)]. Cells with surface-bound POS were washed twice with serum-free DMEM after the 20 °C binding phase before further incubation with DMEM with or without 2 μg/mL protein S (#APP012A, Aniara, West Chester, OH, USA) at 37 °C. All phagocytosis assays were terminated with three washes of phosphate buffered saline (PBS) supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 followed by live imaging or fixation with 4% PFA in PBS.
10
Immunofluorescence Staining of Immune Cells
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Alexa Fluor® 647‐conjugated anti‐mouse CD4 (clone RM4‐5) and anti‐mouse CD8a (clone 53‐6.7) (BioLegend, San Diego, CA, USA, Cat.#: 100530 and 100727, respectively) were used for immunofluorescence at a final concentration of 2.5 μg·mL−1, and eFluor 660 anti‐mouse FOXP3 (clone FJK‐16 s) eBioscience™ (Carlsbad, CA, USA) (Thermo Fisher Scientific, Cat.# 50‐5773‐80) was used at 1 μg·mL−1. eFluor 660 rat IgG2a kappa Isotype Control (eBR2a) (eBioscience™, Thermo Fisher Sci., Cat. # 50‐4321‐80) and Alexa Fluor® 647 Rat IgG2a, κ Isotype Ctrl (BioLegend, Cat. #: 400526) were used as controls for staining specificity. Anti‐Granzyme B (clone D2H2F) Rabbit mAb (Cell Signaling, Danvers, MA, USA, Cat. # 17215S) and rabbit anti‐Ki67 antibody (Abcam, Cambridge, MA, USA, Cat# ab833) were used at 1 : 200 dilution. Goat anti‐rabbit AF488 (Life Technologies, Eugene, OR, USA, Cat. # A‐11008) and Texas Red‐X (Thermo Fisher Cat. # T6391) secondary antibodies were used at 1 : 500 dilution in blocking buffer.
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