Formalin-fixed paraffin sections (6-μm thickness) of IH surgical specimens were dewaxed in xylene and rehydrated in an ethanol series followed by PBS. For placenta sections, after dewaxing and rehydration, placenta sections were placed in 15% glacial acetic acid for 15 minutes to inactivate endogenous alkaline phosphatase (69 (link)) followed by two 5-minutes washes in PBS, before proteinase K treatment. ISH, with 5′, 3′ digoxigenin-labeled locked nucleic acid probes (LNA) for hsa-miR-126a-3p, hsa-miR-517a/c and a scrambled negative control probe (Exiqon), was performed using an Exiqon ISH optimization kit according to the manufacturer’s instructions. All probes were used at a concentration of 40 nM. Hybridization and posthybridization high-temperature washes were performed at 55°C. Probes were detected using alkaline phosphatase–conjugated sheep anti-digoxigenin Fab fragments and NBT/BCIP (both from Roche). Sections were examined and images were collected using a Leica DM4000B microscope and Leica Application Suite software, v4.5.
Ish optimization kit
Manufactured by Qiagen
Sourced in Denmark
The ISH optimization kit is a laboratory equipment product designed to facilitate and optimize in-situ hybridization (ISH) procedures. It provides the necessary components and reagents to support the successful execution of ISH experiments.
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Lab products found in correlation
8 protocols using ish optimization kit
1
In-situ hybridization of placental miRNAs
2
RNA-FISH Visualization in Tissue Sections
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The RNA-FISH assay was performed with the ISH Optimization Kit from Exiqon (Vedbaek, Denmark) as described (Elmen et al., 2008 (link)). Briefly, the mucosa was fixed with 4% fresh paraformaldehyde overnight and embedded in paraffin for sections. The slides were deparaffinized and then incubated with proteinase-K. After washes with PBS, the slides were dehydrated and hybridized with 25 nM fluorescent locked nucleic acid (LNA) probe for 1 h at 60°C. The slides were washed with saline sodium citrate buffer and PBS, covered with coverslides, and then processed using a Zeiss confocal microscope (Zeiss, Jena, Germany).
3
Fluorescent RNA-FISH Assay Protocol
4
RNA-FISH Localization in Tissue Sections
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RNA-FISH was performed with the ISH optimization kit from Exiqon (catalog 90000, Vedbaek) as described (13 (link), 16 (link)). Briefly, the mucosa was fixed with 4% fresh paraformaldehyde (PFA) overnight and embedded in paraffin for sectioning. The slides were deparaffinized and then incubated with proteinase-K. After washes with PBS, the slides were dehydrated and hybridized with 25 nM fluorescent LNA-probe for 1 hour at 60°C. The slides were washed with SSC buffers and PBS, covered with cover slides, and then processed using a Zeiss confocal microscope.
5
Quantifying Microvessel Density and Biomarker Expression in NPC Tumors
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For quantification of microvessel density (MVD) in NPC tumor samples, the number of blood vessels staining positive for CD31 (1:100 dilution, Abcam, Cambridge, MA, USA) was recorded in ten random fields at 200 magnification. The expression of MDK in NPC tumor samples was examined with IHC as previously described [16 (link)]. The antibody was purchased from Abcam (rabbit monoclonal anti-Midkine, Abcam). In situ detection of miR-9 on formalin-fixed paraffin-embedded samples was essentially performed with a miRCURY LNA™ miR-9 detection probe, using an ISH optimization kit (Exiqon, Vedbaek, Denmark) [10 (link)]. A scramble-miR probe (Exiqon) was performed as negative control.
6
Quantitative Analysis of miR-31 and RhoA in Gastric Cancer
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In situ hybridization of miR-31 for formalin-fixed and paraffin-embedded GC and pericarcinous tissue samples was performed using a miRCURY LNA™ miR-31 detection probe via ISH optimization kit (Exiqon, Vedbaek). Scramble-miR probes were used as negative controls.
Immunohistochemical analysis of RhoA in tissue samples was carried out using the EnVision two-step method, as previously described [16 (link)]. Rabbit polyclonal anti-RhoA antibody (Zhongshan Biology Company, Beijing) was used as the first antibody and was diluted 100 times. The avidin-biotin-peroxidase anti-goat antibody was used as the second antibody (5 μg/mL, Vecor Laboratories, CA, USA). RhoA was stained a brown color and results were quantified as follows: − (stained cells <5%), + (stained cells 5–25%), ++ (stained cells 25–75%), +++ (stained cells >75%) [17 (link)]. Results from immunohistochemistry were independently assessed by two observers.
Immunohistochemical analysis of RhoA in tissue samples was carried out using the EnVision two-step method, as previously described [16 (link)]. Rabbit polyclonal anti-RhoA antibody (Zhongshan Biology Company, Beijing) was used as the first antibody and was diluted 100 times. The avidin-biotin-peroxidase anti-goat antibody was used as the second antibody (5 μg/mL, Vecor Laboratories, CA, USA). RhoA was stained a brown color and results were quantified as follows: − (stained cells <5%), + (stained cells 5–25%), ++ (stained cells 25–75%), +++ (stained cells >75%) [17 (link)]. Results from immunohistochemistry were independently assessed by two observers.
7
Detection of Exogenous antimiR-145 in FFPE Mouse Lung
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FFPE blocks of mouse lung were cut into sections 5 μm thick, and ISH was used to detect distribution of exogenously administered antimiR-145. Custom DIG-labeled LNA probes (Exiqon) and ISH optimization kits were used according to the manufacturer’s instructions (Exiqon, #90-007). The LNA double-DIG-labeled antimir-145 probe was diluted to 60 nM (antimiR-145 and nontargeting oligonucleotides). Anti-DIG alkaline phosphatase (AP) antibody (#11093274910, Sigma-Aldrich, St. Louis, MO, USA) was used to visualize antimiR-145 (Figure 1 B). Slides were counterstained with Nuclear Fast Red prior to mounting coverslips.
8
Angiogenesis and FBXW7 in NPC
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Paraffin-embedded NPC tissues were cut into 5 μm sections. The sections were made into slides and then immunostained for CD31 and FBXW7. Immunohistochemical staining for CD31, an endothelial cell marker, was performed to identify angiogenesis in the NPC tissues. The number of CD31-positive vessels (1:100 dilution, Abcam, Cambridge, UK) was recorded in 10 random fields of view at 200× magnification. In accordance with the aforementioned, the expression of FBXW7 was examined in NPC tumor samples with the use of immunohistochemical staining. All antibodies were purchased from Abcam. The in-situ detection of miR-144 was identified in formalin-fixed paraffin-embedded samples using ISH optimization kits (Exiqon, Vedbaek, Denmark) with miRCURY LNA miR-144 probe, and scramble-miR probe (Exiqon) was used as a NC.
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