Coverslips prepared for RNA fluorescence in situ hybridization (FISH) staining were adapted from previously published protocols (Byron et al., 2013 (link); Jiang et al., 2013 (link)). In brief, coverslips were coated with appropriate attachment protein solution and seeded with cells. After cell attachment, coverslips were fixed with 4% paraformaldehyde in 1X phosphate buffer saline solution (PBS) for 10 min then extracted with 0.5% Triton X- in 10mM vanadyl ribonuclease complex (VRC) for 3 min and stored in cold 1X PBS or 70% ethanol. For IF, coverslips were fixed as described for RNA FISH or with 100% cold methanol for 10 min. For RNA FISH, we used a Stellaris probe (Biosearch Technologies, SMF-2038-1) to detect XIST RNA. The APP probe was generated using a BAC from BACPAC Resources (RP11-910G8) and labeled via nick translation with Digoxigenin-dUTP (Roche). To dual stain for protein and RNA, RNAsin (Promega) was added to the primary and secondary antibody stains as described by Byron et al. (2013) (link). To assess proliferation BrdU was incubated for two hours in day 10 endothelial cells and fixed as described above. Coverslips were incubated in 70% formamide in 2x SSC for 5 min followed by a 5 min dehydration step in 70% and 100% cold ethanol, then stained for IF. All antibodies used are listed in the Key resources table .
Stellaris probe
Manufactured by Biosearch Technologies
Sourced in United States
Stellaris probes are fluorescent-labeled oligonucleotide probes designed for the detection and quantification of RNA targets. The probes are used in Stellaris RNA FISH (Fluorescence In Situ Hybridization) assays to visualize and analyze the expression and localization of target RNA molecules within cells.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (3 mentions)
Digoxigenin dutp, by Roche (2 mentions)
Rnasin, by Promega (2 mentions)
Quasar 670, by Biosearch Technologies (2 mentions)
Quasar 570, by Biosearch Technologies (2 mentions)
Purified formaldehyde, by Electron Microscopy Sciences (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Stellaris rna fish hybridization buffer, by Biosearch Technologies (1 mentions)
Lsm 780 microscope, by Zeiss (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Wash buffer a, by Biosearch Technologies (1 mentions)
D8906, by Merck Group (1 mentions)
Vanadyl robonucleoside complex, by New England Biolabs (1 mentions)
Catalase, by Merck Group (1 mentions)
G2133 10ku, by Merck Group (1 mentions)
Sp6 t7 transcription kit, by Roche (1 mentions)
17 protocols using stellaris probe
1
RNA FISH and Immunofluorescence Staining
2
RNA FISH and Immunofluorescence Staining
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Coverslips prepared for RNA fluorescence in situ hybridization (FISH) staining were adapted from previously published protocols (Byron et al., 2013 (link); Jiang et al., 2013 (link)). In brief, coverslips were coated with appropriate attachment protein solution and seeded with cells. After cell attachment, coverslips were fixed with 4% paraformaldehyde in 1X phosphate buffer saline solution (PBS) for 10 min then extracted with 0.5% Triton X- in 10mM vanadyl ribonuclease complex (VRC) for 3 min and stored in cold 1X PBS or 70% ethanol. For IF, coverslips were fixed as described for RNA FISH or with 100% cold methanol for 10 min. For RNA FISH, we used a Stellaris probe (Biosearch Technologies, SMF-2038-1) to detect XIST RNA. The APP probe was generated using a BAC from BACPAC Resources (RP11-910G8) and labeled via nick translation with Digoxigenin-dUTP (Roche). To dual stain for protein and RNA, RNAsin (Promega) was added to the primary and secondary antibody stains as described by Byron et al. (2013) (link). To assess proliferation BrdU was incubated for two hours in day 10 endothelial cells and fixed as described above. Coverslips were incubated in 70% formamide in 2x SSC for 5 min followed by a 5 min dehydration step in 70% and 100% cold ethanol, then stained for IF. All antibodies used are listed in the Key resources table .
3
Quantifying fsSox2 Transcription by smFISH
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Briefly, 70–80% confluent 1 × 106 HeLa (ATCC® CCL‐2™) cells were electroporated with 2 μg of construct using the Amaxa® Cell Line Nucleofector® Kit R using program I‐013, and cultured for 48 h in LabTek v1 glass chambers. smFISH was performed using Biosearch Technologies Stellaris® probes, as described previously (Hacisuleyman et al, 2016 ). RNA probes targeting and tiling the fsSox2 exon were conjugated to Quasar 570. Nuclei were visualized with 4,6‐diamidino‐2‐phenylindole (DAPI). Images were obtained using the Zeiss Cell Observer Live Cell microscope at the Harvard Center for Biological Imaging. For each field of view, at least 40 slices (each plane: 0.24 μm) were imaged, and z‐stacks were merged with maximum intensity projections (MIP). fsSox2 foci were computationally identified using the spot counting software StarSearch. To ensure robustness, the analysis was blinded and the person counting the spots did not know the identity of the samples. For each construct, fsSox2 foci within at least 150 cells were counted in biological duplicate.
4
Visualization of Active Transcription Sites
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Active transcription sites were visualized by fluorescence in situ hybridization (FISH) using sets of 48 20-mer oligonucleotides (Stellaris®-probes; LGC Biosearch Technologies, Petaluma, CA, USA). One set was designed to hybridize with intronic sequences of MYH7-pre-mRNA and each oligonucleotide was labeled with one Cy5-like fluorophore (Quasar 670, LGC Biosearch Technologies). The other set was designed to hybridize with exonic sequences of MYH7-mRNA and labeled with a Cy3-like fluorophore (Quasar 570; LGC Biosearch Technologies). Both probe sets were custom made (Stellaris® Probe Designer). Following hybridization, active transcription sites were taken as bright spots inside nuclei of cardiomyocytes showing both fluorescence signals. Further details are described in Supplementary Material.
5
Single-Molecule FISH Using Stellaris Probes
6
Single-Molecule RNA FISH Assay
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smRNA FISH was performed using Stellaris probes and the Stellaris protocol (Biosearch Technologies). Probes were designed with the Stellaris Probe Designer using 1.5 to ∼2.5 kb of the 5′ end of the coding sequence. FISH probe sets for each gene consisted of ∼33 20mer DNA oligonucleotides, complementary to the target RNA. A probe set for MS2 repeats consisted of eight 20mer oligonucleotides. Probe sets are listed in Table 1 .
7
Stellaris smFISH Experiments Protocol
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Stellaris smFISH experiments were performed as previously described (64 (link)) and following the manufacturer's protocol with a few modifications. Following fixation with 4% paraformaldehyde (Electron Microscopy Sciences), coverslips were methanol treated for 30 min in −20°C and rehydrated twice in 1X PBS, each time washing for 5 min. Cells were washed twice in 2× SSC buffer (150 mM NaCl, 15 mM NaCitrate, pH 7.1) with 10% formamide. 100 μl of hybridization probe (100 mg/ml dextran sulfate, 10–15% formamide, 2× SSC) containing 125 nM Stellaris probes (LGC Biosearch Technologies) was added to the coverslips and incubated for 48 h. Subsequently, the coverslips were washed three times with 2× SSC buffer with 10–15% formamide and mounted onto coverslips using DAPI Fluoromount-G stain mounting solution (Southern Biotech). Cells were imaged and quantified as previously described (64 (link)).
8
Single-Molecule FISH Analysis of C. elegans Gonads
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smFISH analysis was performed on day 1 adult male and hermaphrodite gonads dissected from him-8(e1489) worms as in ref. 56 . Biosearch Technologies designed, synthesized, and labeled the Stellaris probes. The pie-1, par-6, and nos-2 probes were coupled to Quasar 670, the mex-3 probe was coupled to Cal Fluor Red 590, and the cpg-2 probe was coupled to Cal Fluor Red 610. Each probe was resuspended in 250 μL of TE Buffer pH 8.0 and then further diluted to 1:30 for hybridization. The microscope and its settings are as in ref. 52 (link). Fig. 3 and Supplementary Fig. 6 contain montages generated by splicing together contiguous images acquired with identical settings. Male and hermaphrodite pairs used identical confocal settings, with the exposure optimized for visualizing the male gonads. All images were processed identically with ImageJ and Adobe Illustrator. For both oogenic and male germlines, 10–60 gonads were visually examined under the microscope and at least 3 germlines were imaged in 1–3 separate experiment(s).
9
RNA FISH Protocol for Cellular Localization
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Cells were fixed in 4% purified formaldehyde (Electron Microscopy Sciences) in ribonuclease (RNase)-free PBS for 15 min at room temperature and then permeabilized with 70% ethanol in RNase-free water at 4°C for a minimum of 1 hour [21 (link)]. Cells were washed in 1 ml of wash buffer (2x saline sodium citrate [SSC] plus 10% formamide) followed by overnight hybridization with RNA FISH probes at 37°C (Stellaris® probes; LGC Biosearch Technologies) in Stellaris® RNA FISH hybridization buffer (SMF-HB1-10), followed by one change in wash buffer (30 min at 37°C), and a second wash in buffer containing 1μg/ml DAPI (10 min at 37°C) [21 (link)]. Vectashield (Vector Laboratories) was used as the mounting medium. All probes were custom designed, synthesized, and labeled with either Quasar 570® or Quasar 670® dyes by LGC Biosearch Technologies.
10
Quantitative RNA FISH analysis of FLO11 expression
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RNA FISH was conducted essential as described previously (McIsaac et al. 2013 (link)) using Stellaris probes from Biosearch Technologies. Thirty-five 20mer Quasar 670 probes covering the stretch +9 to +708 of FLO11 were used for detection of FLO11 mRNA, whereas 35 20mer Quasar 570 probes covering the stretch +2 to +701 of ACT1 were used as positive hybridization control. Precultures were grown overnight at 30° in synthetic medium. Cells were subsequently inoculated into synthetic medium to OD600nm 0.1 for 20 hr before fixation. Cells were fixated with 3% paraformaldehyde for 30 min at 30° followed by 4-hr incubation at 5°. Expression of FLO11 was subsequently recorded with a Zeiss LSM780 microscope. Cells were counted as recordable if they were labeled with the ACT1 probe. All ACT1 positive cells were subsequently recorded for their expression of FLO11 mRNA by counting cells with one or more red foci as positive for FLO11 mRNA. A total of 1329 wild-type cells, 1167 sfl1 cells, and 542 flo11 cells were counted as blinded samples. The flo11 cells served a negative control for FLO11 mRNA expression. None of the flo11 cells showed any signal for FLO11 mRNA labeling.
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