In Situ Hybridization (ISH) was conducted using an In situ hybridization kit (Boster, China). Tissue sections were processed to de-paraffinization, dehydration and digested with pepsin. Subsequently, sections were incubated with FAM225A-specific probe at 50°C overnight. Sections were further incubated with biotinylated anti-Digoxin (Boster, China), stained with biotinylated peroxidase, and visualized with 3,3'-diaminobenzidine substrate.
In situ hybridization kit
Manufactured by Boster Bio
Sourced in China
The in situ hybridization kit is a laboratory tool used to detect and localize specific nucleic acid sequences within intact cells or tissue sections. It allows the visualization and analysis of gene expression patterns and chromosomal structures.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated anti digoxin, by Boster Bio (2 mentions)
Bm purple ap substrate, by Roche (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluorescence secondary antibody, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscopy, by PerkinElmer (1 mentions)
Dapi karyotyping kit, by Genmed Scientifics (1 mentions)
Avidin biotin peroxidase complex abc kit, by Vector Laboratories (1 mentions)
Anti c myc, by Merck Group (1 mentions)
Hematoxylin, by Beyotime (1 mentions)
Anti ccnd1, by Santa Cruz Biotechnology (1 mentions)
Anti ps473 akt, by Bioworld Technology (1 mentions)
12 protocols using in situ hybridization kit
1
In Situ Hybridization for FAM225A Detection
2
In-situ Hybridization of Glioma miRNA
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Using sense locked nucleic acid (LNA)-modified oligonucleotide probes, in situ hybridization was performed on paraffin-embedded sections (4μm thickness) of glioma specimens with an in situ hybridization kit (Boster Biological Technology, Ltd., Wuhan, China). After processing with 3% H2O2, sections were treated with proteinase K (2μg/ml) at 37°C for 30min, washed, and prehybridized for 2h at 37°C. Hybridization with digoxygenin (DIG)-labeled miRCURY LNA probes (probe sense: 5′-ACGCAGAGCCCGAAAGCCCCCAGT-3′) was performed overnight at 37 °C. Slides were then washed at 37 °C and incubated with alkaline phosphatase–conjugated sheep anti-DIG Fab fragments for 1h at room temperature. Staining was visualized by adding BM purple AP substrate (Roche, Basel, Switzerland) according to the manufacturer’s instructions.
3
In Situ Detection of miR-340 Expression
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Specimens were fixed in 10% formaldehyde, embedded by paraffin, and cut into 3 μm sections. Sections were transferred onto a special glass slide that was pretreated with 10% polylysine. The protocol was carried out in accordance with the manufacturer’s instructions of in situ hybridization kit (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). After digoxin-labeled miR-340 probe (Exiqon, Denmark) was dripped in, sections were hybridized at a constant temperature of 52°C for 16 h and then left in a warm-bath with biotinylated mouse anti-digoxin antibody at 37°C for 60 min followed by incubation in strept avidin–biotin complex (SABC). Next, diaminobenzidine (DAB) was utilized to develop color. The results were scored by two pathologists independently. Cells with blue-stained cytoplasm were considered positive. Five fields were randomly selected from each section under a light microscope (200×). Through observation, the percentage of positive cells was calculated. Specimens were considered negative if the percentage of positive cells was less than 5% and positive if the percentage was more than or equal to 5%.
4
In Situ Detection of miR-503-5p in Prostate Cancer
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Oligonucleotide probes modified by locked nucleic acid (LNA) were used for in situ hybridization with in situ hybridization kit (Boster, China). In situ hybridization was used to detect the expression of miR-503-5p in prostate cancer specimens.The LNA/DNA oligonucleotide contains locked nucleic acids at 8 consecutive centrally located bases and has the following sequence: LNA-miR-503-5p, 5’-TCAACATCAGTCTGATAAGCTA-3’. DAPI karyotype kit (GenMed, USA) was used for reverse staining, and FluoView confocal laser scanning microscope FV1000 was used for observation.
5
In Situ Hybridization of circ_0016070
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In situ hybridization staining was performed using an in situ hybridization kit (Boster, China). PH tissue sections were deparaffinized, dehydrated, and pepsin‐digested. Samples were incubated with circ_0016070‐specific probe overnight at 50 °C and further incubated with biotinylated antidigoxin (Boster, China), stained with biotinylated peroxidase, and developed with 3,3′‐diaminobenzidine substrate. Then, the sections were observed by using a light microscope.
6
In Situ Hybridization for miR-372 Expression
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The in situ hybridization kit was purchased from Boster Company (Wuhan, China) and used according to the manufacturer’s instructions. Briefly, the tissue slides were hybridized with 20 ul of 5′-digoxigenin (DIG) LNA-modified-miR-372-3p. The nucleic acid sequence is 5′-ACGCTCAAATGTCGCAGCACTTT-3′. Results were independently scored by two experienced pathologists. The scoring of positive tumor cells was as follows: 0 (0%), 1 (1–10%), 2 (11–50%) and 3 (>50%). The staining intensity was visually scored as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The miR-372 expression score was calculated from the value of percent positivity score multiplied by the staining intensity score. This value ranged from 0 to 12, and the tumors were classified as follows: negative (−), score 0; lower expression (1+), score 1–4; moderate expression (2+), score 5–8; and strong expression (3+), score 9–12. In situ hybridization miR-372 staining was grouped into two categories: low expression (0/1+) and high expression (2+/3+).
7
Profiling circCDYL2 Expression by FISH and ISH
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Fluorescence in situ hybridization (FISH) and in situ hybridization (ISH) analyses were conducted using the in situ hybridization kit (BOSTER, Wuhan, China) according to the manufacturer’s instructions. Dig-labeled probes (Sangon Biotech, Shanghai, China) were used to detect circCDYL2 expression in nasopharyngeal carcinoma cells or clinical tissue samples. FISH employed fluorescence secondary antibody (Invitrogen, CA, USA), while ISH used DAB staining reagents (Meixin Biology, Fujian, China). Cell nuclei were counterstained with DAPI (Invitrogen, CA, USA, for FISH) or hematoxylin (for ISH). FISH slides were observed and photographed using confocal laser scanning microscopy (Perkin Elmer, MA, USA). The ISH slides were independently evaluated by two pathologists and analyzed using a semi-quantitative integral method. The sequences of circCDYL2 probes used in this study are provided in Supplemental Table 3 .
8
In Situ Hybridization of miR-141 in Glioma
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In situ hybridisation (ISH) was performed with an in situ hybridization kit (Boster Biological Technology, Ltd., Wuhan, China). The glioma tissue microarrays were deproteinated, and then prehybridized for 2 h in hybridization liquid in a humidified chamber (50% formamide, 5 × SSC). MiR-141 probes were added to the sections on the microarray and incubated overnight at 40°C in a water bath. The anti-digoxigenin-rhodamine and streptavidin-FITC solution was added and incubated for 2 h at room temperature in the dark. Nuclei were counterstained with a DAPI karyotyping kit (Genmed, USA). Sections were sealed and detected under a fluorescence microscope with an OptiGrid system and analyzed by IPP6.1 (Olympus, Tokyo, Japan).
9
PCAT-14 Expression Quantification
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The in situ hybridization kit was purchased from Boster Company (Wuhan, China) and used according to the manufacturer's instructions. Briefly, the tissue slides were hybridized with 20 ul of 5′-digoxigenin (DIG) LNA-modified-PCAT-14–3p. Results were independently scored by two experienced pathologists. The scoring of positive tumor cells was as follows: 0 (0%), 1 (1–10%), 2 (11–50%), 3 (51–80%) and 4 (>80%). The staining intensity was visually scored as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The PCAT-14 expression score was calculated from the value of percent positivity score multiplied by the staining intensity score. This value ranged from 0 to 12, and the tumors were classified as follows: negative (−), score 0; lower expression (1+), score 1–4; moderate expression (2+), score 5–8; and strong expression (3+), score 9–12. In situ hybridization PCAT-14 staining was grouped into two categories: low expression (0/1+) and high expression (2+/3+).
10
Detecting Cardiac Hormone Receptor mRNAs
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