Custom Stellaris® FISH probes were designed against GFP or smo mRNA by utilizing the Stellaris® FISH probe designer Biosearch Technologies, Inc. (Petaluma, CA, USA). Wing discs were hybridized with GFP and smo Stellaris® FISH probe sets labelled with Quasar 670 and Quasar 570 (Biosearch Technologies, Inc.), respectively, following the manufacture's protocol using 250 nM per probe set. Briefly, wing discs from up‐crawling third‐instar larvae were dissected in PBS, fixed with 4% paraformaldehyde for 40 min on ice and then washed twice in PBS for 5 min. For permeabilization, wing discs were incubated overnight in 70% ethanol at 4°C. After washing for 10 min with wash buffer A (Biosearch Technologies, #SMF‐WA1‐60), wing discs were incubated overnight with Stellaris® FISH probes in hybridization buffer (Biosearch Technologies, #SMF‐HB1‐10) at 37°C. Subsequently, wing discs were washed in wash buffer A for 30 min at 37°C and in wash buffer B (Biosearch Technologies, #SMF‐WA1‐20) for 30 min at room temperature. Wing discs were finally mounted in VectaShield®.
Stellaris fish probe
Manufactured by Biosearch Technologies
Sourced in United States
Stellaris FISH probes are fluorescently labeled DNA or RNA sequences designed for in situ hybridization. They are used to detect and visualize specific nucleic acid targets within cells or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m, by Zeiss (4 mentions)
Stellaris rna fish hybridization buffer, by Biosearch Technologies (3 mentions)
Quasar 570, by Biosearch Technologies (3 mentions)
Ribonucleoside vanadyl complex, by New England Biolabs (2 mentions)
Observer z1 microscope, by Zeiss (2 mentions)
Stellaris rna fish wash buffer a, by Biosearch Technologies (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Quasar 570 dye, by Biosearch Technologies (2 mentions)
Stellaris rna fish probe, by Biosearch Technologies (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Quasar 670, by Biosearch Technologies (2 mentions)
Dextran sulfate, by Merck Group (1 mentions)
Formamide, by Thermo Fisher Scientific (1 mentions)
Formamide, by Merck Group (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Quasar 670 dye, by Biosearch Technologies (1 mentions)
Cs 25r, by Warner Instruments (1 mentions)
Round cover glasses, by Avantor (1 mentions)
24 protocols using stellaris fish probe
1
Stellaris® FISH Probe Labeling of Drosophila Wing Discs
2
Visualizing lncRNA Expression in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LncRNA probes were designed and synthesized by Biosearch Technologies. As a cytoplasmic positive control we used the protein-coding gene GAPDH, for which Stellaris FISH Probes (Biosearch Technologies) were commercially available. RNA FISH experiments were performed on HeLa cells following Stellaris RNA FISH protocol for adherent cells. Imaging was performed using an inverted fluorescent microscope.
3
Visualization of NEAT1 Long Isoform in HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLaGFP-NONO cells, uninduced or expressing GFP-NONO, were seeded at approximately 70% confluency on the day prior to fixation, then fixed for 10 min in 4% paraformaldehyde (in PBS), and washed once with PBS. Cells were permeabilized in 70% ethanol for at least 1 h at 4 °C and washed once with wash buffer for 5 min. Wash buffer was prepared with 10% formamide (Ambion) in 2× SSC (contains 0.3 M of NaCl and 30 mM of sodium citrate). Hybridization buffer was prepared with 100 mg/ml dextran sulfate (Sigma) and 10% formamide in 2× SSC. A hybridization mixture was prepared using 125 nM of Stellaris FISH Probes (Biosearch Technologies, Inc) targeted to the long isoform of human NEAT1 labeled with Quasar 570 Dye (SMF-2037-1, Biosearch), made to a final concentration of 100 mg/ml dextran sulfate, 10% formamide, and 2× SSC. Each coverslip was transferred onto a 50 μl drop of hybridization mixture, cells side down, incubated in a sealed, dark humidified chamber overnight at 37 °C. Coverslips were then rinsed with wash buffer for 30 min at 37 °C in the dark, counterstained with DAPI (5 ng/ml in wash buffer) for 30 min at 37 °C in the dark, and finally rinsed in 2× SSC. Coverslips were mounted onto microscopic slides using Vectashield mounting medium.
4
Spatial Colocalization of MYC and PVT1 Transcripts
5
NEAT1 lncRNA Localization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After fixation, cells were stored in 70% ethanol at 4 °C overnight for cell permeabilization. Commercially available probe sets against the NEAT1 5’ segment and middle segment (Stellaris FISH probes, NEAT1 5’ segment with Quasar 670 dye, and NEAT1 middle segment with Quasar 570 dye, Biosearch Technologies, Teddington, UK) and wash and hybridization buffers (Biosearch Technologies) were used according to the manufacturer’s instructions. Hybridization was performed at 37 °C for 5 h. Coverslips were mounted with Prolong Diamond (Invitrogen, Waltham, MA, USA). Slides were stored at −20 °C.
6
Transcriptional Bursting of Beta-Actin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We designed 25 DNA FISH probes (Stellaris FISH Probes from LGC Biosearch Technologies, Novato, CA) labeled with Fluorescein (FITC), targeting intron 1 of beta-actin gene. To investigate transcriptional bursting of beta-actin under serum-stimulation, wild type MEF and endogenous Dendra2-Pol II MEF cells were grown on 25 mm round cover glasses (from Warner Instruments, CS-25R) for and starved in serum/FBS free DMEM for 12 hours. The cell were then serum-stimulated by exchanging the starving medium DMEM supplemented with 10% FBS. Cells were fixed with 4% paraformaldehyde, at time points corresponding to 5 min,10 min, 15 min, 20 min, 25 min and 30 min after serum-stimulation. For probe hybridization, we followed the Stellaris protocol. After fixation, to permeabilize the cells, the cells were incubated in 70% ethanol for 1 hr at 4 °C. After removing the ethanol with the Stellaris washing buffer, cells were incubated with 125 nM probe solution overnight at 37 °C in a humidified chamber. All sample dishes were imaged at same optical conditions and the same days for illumination consistency.
7
RNA FISH Analysis of NEAT1 Isoforms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA FISH was performed using Stellaris FISH probes (Biosearch Technologies) for human NEAT1: SMF-2036-1 for NEAT1_5 and VSMF-2251-5 for NEAT1_m. FISH was performed according to the manufacturer’s protocol. Briefly, cells were grown on slides (round cover glasses; VWR), fixed in 4% paraformaldehyde (PFA), and permeabilized in 70% EtOH over night at 4°C. Cells can be stained within the following 2 weeks maximum. Cells were washed twice in PBS and incubated for 5 min in FISH washing buffer [2× standard saline citrate (SSC) and 10% formamide]. Hybridization of FISH probes was carried out overnight at 37°C in 2× SSC, 10% formamide, and 10% dextran, in a dark humid chamber. After three washes with FISH washing buffer, slides were mounted in ProLong Gold Antifade containing 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) and images acquired on a confocal microscope Nikon C2. Imaging panels were prepared using Imaris 7.2.3 and ImageJ [plugins such as Interactive three-dimensional (3D) surface plot and JACoP (Just Another Colocalization Plugin) were used, respectively, to produce the 3D plots to show colocalization and to quantify fluoresce and signal colocalization]. In Fig. 2I , 25 MCF-7 cells randomly selected cells from three biological replicates were used for the quantification.
8
Quantitative RNA FISH Imaging and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stellaris RNA FISH probes targeting FOS, JUN, and XIST were designed and generated by Biosearch Technologies. RNA FISH was performed according to Stellaris’ RNA FISH protocol (Biosearch Technologies). Briefly, cells were grown on 12-mm round coverslips in a 12-well plate, fixed by 3.7% formaldehyde for 10 min, and permeabilized by 70% ethanol for at least 1 hour. Cells were then washed with Wash Buffer A (Biosearch Technologies) and hybridized with 50 μl of hybridization buffer (Biosearch Technologies) containing Stellaris FISH probes for at least 4 hours at 37°C in a humidified chamber. Coverslips were mounted using VECTASHIELD Mounting Medium with DAPI. Images were acquired using a Zeiss LSM 700 confocal microscope with 63×, 1.4 NA oil immersion objective lens using Zen Light Edition acquisition software and CCD camera. Images were postprocessed using Zen Light Edition and FIJI. The distribution fitting of RNA FISH foci intensity data was analyzed using custom-written MATLAB (R2016a) scripts. Results shown are representative of at least three biological replicates.
9
LINC02273 RNA Localization by FISH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LINC02273 RNA FISH was performed using Stellaris FISH probes as listed in Additional file 1 : Table S11 (Biosearch Technologies) according to the manufacturer’s protocol. In brief, MDA-MB-231 cells were grown on coverslips in a 24-well culture plate. Cells were fixed with 4% (w/v) paraformaldehyde in 1 × PBS for 10 min. Fixed cells were permeabilized with 70% ethanol for 1 h at 4 °C. The coverslips were washed one time with Buffer A (Biosearch Technologies) for 5 min at room temperature and incubated with Stellaris RNA FISH Hybridization Buffer (Biosearch Technologies) containing 10% formamide and 250 nM of the LINC02273-specific Stellaris probe labeled with CAL Fluor Red 590 in the dark at 37 °C overnight. The coverslips were then washed once with Wash Buffer A and once with Wash Buffer A containing 0.1 mg/ml DAPI, both washes in the dark at 37 °C for 30 min and mounted onto microscope slides using VECTASHIELD Antifade Mounting Medium (Vector Laboratories). Images were acquired with Leica confocal microscope.
10
Custom FISH Probes for MALAT1 Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A set of custom Stellaris® FISH probes comprising 17 single-labeled oligonucleotides designed for MALAT1 were purchased from Biosearch Technologies. TALAM1 probe was generated via nick-translation cDNA kit (Abbott Molecular, USA), using a pGEM-Teasy construct containing the 5′ 1 kb unique region of TALAM1 as template. For RNA/DNA FISH, DNA FISH probe was generated via nick translation from BAC clone RP11–1104L6. For dual RNA FISH, the fixation and hybridization of HeLa cells were performed as described previously (20 (link)). For RNA/DNA FISH, the RNA FISH probe against TALAM1 was labeled with digoxigenin-11-dUTP using DIG nick translation mix (Roche), and fixation and hybridization of HeLa cells were performed as described previously (21 ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!