RNA fluorescence in situ hybridization (FISH) was performed using the Fluorescent in situ Hybridization Kit (lnc1CM001, RiboBio) according to the manufacturer’s instructions. Cy3-labeled probes targeting circRFWD3, 5FAM-labeled miR-27a/27b, and Cy3-labeled probes targeting PPARγ were synthesized by RiboBio (Guangzhou, China). Fluorescence was recorded with a confocal laser scanning microscope (FV3000, OLYMPUS).
Cy3 labeled probes
Manufactured by RiboBio
Sourced in China
Cy3-labeled probes are fluorescent nucleic acid probes that are labeled with the Cy3 dye. Cy3 is a cyanine dye that emits in the orange-red region of the visible spectrum, making it useful for a variety of applications such as fluorescence in situ hybridization (FISH) and real-time PCR. The core function of Cy3-labeled probes is to provide a fluorescent signal for the detection and visualization of target sequences.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescent in situ hybridization kit, by RiboBio (7 mentions)
Airyscan, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Bx63 microscope, by Olympus (1 mentions)
Bx61 microscope, by Olympus (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg h l, by ZSGB-BIO (1 mentions)
Anti ki67, by Abcam (1 mentions)
U6 rna, by RiboBio (1 mentions)
A1si laser scanning confocal microscope, by Nikon (1 mentions)
Fv3000, by Olympus (1 mentions)
Aisi laser scanning confocal microscope, by Nikon (1 mentions)
Fish kit, by RiboBio (1 mentions)
11 protocols using cy3 labeled probes
1
Visualizing circRFWD3, miR-27a/27b, and PPARγ
2
circSLC38A8 Expression Detection Protocol
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Cy3-labeled probes targeting the back-splice junction of circSLC38A8 was designed by RiboBio (Guangzhou, China), and probe sequences are listed in Table S3. The kit was purchased from RiboBio, and the FISH assay was conducted as previously described [24] .
3
RNA FISH for circATP2B4 and SREBF1 in Ovarian Cancer
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RNA FISH was performed using the Fluorescent In Situ Hybridization Kit (C10910, RiboBio) according to the manufacturer's instructions. Cy3-labeled probes targeting circATP2B4 and FITC-labeled SREBF1 probes (see Supplementary Table S3) were synthesized by RiboBio Technology Co. Ltd. Fluorescence was excited and recorded with a Olympus BX63 Microscope (BX63, Olympus). Fifteen EOC patient FFPE samples were used in the in situ hybridization (ISH) assay, which were diagnosed and tumor tissue surgically removed at Zhejiang University Women's Hospital in 2019 after informed consent by patients and included 10 patients without metastasis and 5 patients with peritoneal metastasis. The protocol of ISH performed on paraffin sections of ovarian cancer tissues or animal tumors was taken from previous literature (25 (link)). Briefly, after dewaxing and rehydration, the sections were digested with pepsin, and then hybridized with the digoxin-labelled nucleotide sequence (Synbio-Tech, China) overnight at 37°C. Next, the sections were incubated with anti-digoxin overnight at 4°C. The sections were stained with DAB and counterstained with hematoxylin. RNA expression excited and recorded with a Olympus BX61 Microscope (BX61, Olympus).
4
Visualizing circNOLC1 and miR-647 via FISH
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CircNOLC1 and miR-647 were captured by Cy3-labeled probes (RiboBio, Guangzhou, China) and Alexa 488-labeled probes (FOCOFISH, Guangzhou, China). FISH experiment was conducted using Fluorescent in situ Hybridization Kit (No. C10910, RiboBio, Guangzhou, China), according to the official guidelines. DAPI was utilized to stain the nuclei. Subsequently, circNOLC1 and miR-647 were observed through a confocal microscope (LSM 880 with Airyscan, Carl Zeiss, Germany).
5
SATB2-AS1 and miR-3678-3p Localization
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SATB2-AS1 and miR-3678-3p were captured by Cy3-labeled probes (RiboBio, Guangzhou, China) and Alexa 488-labeled probes (FOCOFISH, Guangzhou, China). The Fluorescent in situ Hybridization Kit (No. C10910, RiboBio, Guangzhou, China) was applied for FISH, according to the official guidelines. The nuclei were stained by DAPI. Subsequently, SATB2-AS1 and miR-3678-3p signals were detected with a confocal microscope (LSM 880 with Airyscan, Carl Zeiss, Germany).
6
Fluorescent In Situ Hybridization for circStag1
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Cy3-labeled probes for the backsplicing junction of circStag1 (Table S5 ), U6, and 18S were synthesized by RiboBio (RiboBio Biotechnology, Guangzhou, China). The hybridization was performed in a humidification chamber at 37 °C for 16 h. The circStag1 signals were detected using a Fluorescent in Situ Hybridization Kit (RiboBio, Guangzhou, China) following the manufacturer’s instructions41 (link). Fluorescence images were collected using a Leica DMi8 microscope.
7
Immunofluorescence and RNA-FISH Analysis
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MEC-1 and JVM-3 were plated on slides in 48-well plates and incubated at 60°C for IF and RNA-FISH analysis. After fixation and permeabilization, cells were incubated overnight at 4°C with primary diluted antibody anti-Ki67 (#ab15580, Abcam). The secondary antibody Alexa Fluor® 594 - Conjugated Goat anti-Rabbit IgG (H+L) was purchased from ZSGB-BIO, China. Fluorescence were observed using Axio Scope.A1 (Zeiss). FISH assay was performed using Fluorescent In Situ Hybridization Kit (RiboBio, Guangzhou, China) according to the manufacturer’s guidelines. And Cy3-labeled probes from RiboBio, Guangzhou, China were measured by visualized with a confocal microscopy.
8
RNA FISH of CRNDE, 18S, and U6 RNAs
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The Cy3-labeled probes (red fluorescent signal) for RNA FISH of CRNDE, 18S RNA and U6 RNA were synthesized by RiboBio (Guangzhou, China). RNA FISH was performed using fluorescent in situ hybridization kit (RiboBio) according to the manufacturer’s instructions. DAPI was used for counterstaining of nuclei (blue fluorescent signal). The cells were photographed under a fluorescence microscope (OlympusIX81).
9
Fluorescent In Situ Hybridization Protocol
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Cy3-labeled circHIPK3, 18s, and U6 probes were acquired from RiboBio (Guangzhou, China). The signal intensity of cy3-labeled probes was detected by the Fluorescent in situ Hybridization Kit (C10910, RiboBio, China) according to directions of the manufacturer. All fluorescence images were captured using Nikon-A1Si Laser Scanning Confocal Microscope (Nikon, Japan).
10
FISH Analysis of SBF1 in FFPE Placenta
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FISH was performed with formalin-fixed paraffin-embedded (FFPE) placenta specimens from four FGR and four control cases. Multiple Cy3-labeled probes specific to the junction sites of [SBF1circle 50,447,497–50,447,834] were designed and synthesized by RiboBio (China). FISH was performed using a RiboBio kit according to the manufacturer’s instructions with slight modification. In brief, 5-µm-thick FFPE sections were deparaffinized and rehydrated before pretreatment with protease K. The sections and probes were then co-denatured at 75°C for 5 min and hybridized overnight at 37°C. The sections were washed and counterstained with 4,6-diamino-2phenylindole. Fluorescence images of five randomly chosen areas of each slice were taken and evaluated using Fiji software (Schindelin et al., 2012 (link)); the integrated fluorescence density was measured and a mean value was calculated for each sample and compared between groups.
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