The ST samples were subjected to standard overnight fixation in 10% neutral-buffered formalin followed by paraffin embedding and were cut into 10 μm-thick slices. The slides were then deparaffinized in xylene and ethanol, incubated with Proteinase-K for 10 min at 37 °C, dehydrated with gradient ethanol, and hybridized with 1 nM LNA™ U6 snRNA probe and 50 nM double-DOG LNA™ miRNA probe complementary to miR-23b and LNA™ scrambled miRNA probe, respectively (all the probes were purchased from Exiqon, Demnark). After incubating with and removing the blocking solution, the slides were sequentially incubated with anti-DIG reagent and AP substrate (all were procured from Roche, Mannheim, Germany) protecting from light. Finally, KTBT buffer was added to stop the reaction. Nuclear Fast Red (Solarbio, China) was used for counterstaining of the nucleus. The slides were finally observed under light microscopy.
Ap substrate
Manufactured by Roche
Sourced in Germany
AP substrate is a laboratory reagent used as a substrate for alkaline phosphatase (AP) enzyme detection. It provides a colorimetric or chemiluminescent signal when cleaved by the AP enzyme, allowing for the quantification of AP activity in various biological samples and assays.
Automatically generated - may contain errors
Lab products found in correlation
Mircury lna mirna detection probe, by Qiagen (2 mentions)
In vitro transcription kit, by Roche (2 mentions)
Lna u6 snrna probe, by Qiagen (1 mentions)
Nuclear fast red, by Solarbio (1 mentions)
Nuclear fast red solution, by Merck Group (1 mentions)
Mircury lna mirna ish buffer set, by Qiagen (1 mentions)
Hen egg lysozyme, by Merck Group (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Alkaline phosphatase conjugated anti digoxigenin antibody, by Roche (1 mentions)
Nuclear fast red, by Vector Laboratories (1 mentions)
Eukitt, by Merck Group (1 mentions)
7 protocols using ap substrate
1
In Situ Hybridization of miR-23b in Tissue
2
Lateral Flow Assay for SARS-CoV-2 Detection
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LFA was performed using strips of a commercially available nitrocellulose membrane (ab274103) containing a ‘test line’ (T line) of streptavidin able to bind the biotin-conjugated capture antibody which further binds the samples in combination with the detection antibody. The strips also contain a ‘control-line’ (C line) of immobilized anti-mouse antibody, which shows that the test is valid. The capture and the detection mAbs were diluted in Tris-Glycine SDS Running Buffer 1X with BSA 0.1%, along with the samples, the supernatant of VeroE6 cells infected by SARS-CoV-2, and the lysed nasal swabs from individuals negative or positive for COVID-19. For a single strip, a mix of mAbs and sample was prepared, incubated for 5 min, and loaded into a 96-well plate. A strip was inserted into each well, and the mixture was run for 20 min. Strips were then incubated with isotype-specific alkaline phosphatase (AP)-conjugated anti-mouse Ab. The T and the C lines were detected by AP substrate (Roche), following the manufacturer’s instructions.
3
Detecting Endogenous miR-128-3p in Myocardium
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Endogenous mir128-3p in the myocardium was detected with the miRCURY LNA miRNA Detection Probes (339111, Qiagen) on 6 µm paraffin sections. Sample preparation and hybridization were performed using miRCURY LNA miRNA ISH Buffer Set (339450, Qiagen). Briefly, the tissue was permeabilized with proteinase K for subsequent incubation with the double DIG-labelled LNA probe. Following stringent washing and blocking, samples were incubated with sheep anti-DIG AP FAB fragments (11093274910, Sigma). Freshly prepared AP substrate (11697471001, Roche) was applied for 2 hr at 30° C, which was followed by nuclear counterstaining with Nuclear Fast Red solution (N3020, Sigma). Consecutive sections were stained with H and E for morphological comparison. Brightfield images were captured using an Axiovision upright microscope equipped with a Coolsnap ES camera (Photometrics).
4
In Situ Hybridization for Intestinal Markers
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Intestines from mice were flushed with cold PBS and fixed overnight in formalin. Samples were then dehydrated and embedded in paraffin, sectioned at 5 μm and processed to in situ hybridization. The sections were de-waxed, rehydrated, pretreated and hybridized overnight for 12 h at 65 °C with probes. After several times of wash, sections were incubated in blocking solution for 2 h followed by incubation with alkaline phosphatase-conjugated anti-digoxigenin (1:2,000; Roche) overnight at 4 °C. The sections were washed and further incubated with AP substrate (Roche) for appropriate time depending on signal strength. Olfm4 fragment (2–1622) and Pdgfa fragment (313–709) were cloned into EZ-T vector. Probes were then generated through in vitro transcription with Roche in vitro transcription kit.
5
In Situ Hybridization Bacterial DNA Detection
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ISH performed as previous23 (link). In brief, slides were de-paraffinized, rehydrated then treated with 10 mg/mL hen egg lysozyme (Sigma, Oakville, ON, Canada) for 20 minutes followed by treatment with 100 μg/mL proteinase K in buffer for 10 minutes at 37 °C then washing and dehydration. 150 μL of pre-warmed 2 ng/μL double DIG labeled EUB338 (GCTGCCTCCCGTAGGAGT) probe targeting bacterial 16s rDNA or scrambled probe in hybridization buffer (25 mM Tris-HCl, 100 mM NaCl, 0.5% SDS pH 9) was applied to each sample and incubated at 50 °C for 90 minutes (Sigma, Oakville, ON, Canada)42 . Slides were then washed in six rapid changes of 50 °C wash buffer (10 mM Tris, pH 9.0, 1 mM EDTA). The samples were then blocked first for 30 min. with levamisole, then for 1 hr with Odyssey blocking buffer (LiCor, Lincoln, NE, USA). A 1∶200 dilution of AP (Alkaline Phosphatase) conjugated sheep anti-DIG Fab’ fragments (Roche, Mannheim, Germany) was applied to the slides and incubated o/n at 4 °C. The slides were washed 3 times in PBS and incubated for 2 hours in the dark at 30 °C with AP substrate (Roche, Mannheim, Germany), then washed 3 times in PBS, mounted and imaged.
6
In Situ Hybridization Protocol for Olfm4 Gene
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For in situ hybridization experiments, the paraffin sections were de-waxed, rehydrated and hybridized with digoxigenin-labeled probe at 65°C for overnight. After several rounds of wash, sections were placed in blocking solution for 2 h followed by incubating with alkaline phosphatase-conjugated anti-digoxigenin antibody (1:2000; Roche) at 4°C overnight. Then the sections incubated with AP substrate (Roche) after washing several times. Olfm4 probe (2–1622) was generated with in vitro transcription kit (Roche).
7
In Situ Hybridization of miR-200b-3p
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A double digoxigenin-labeled (5′ and 3′) miRCURY LNA miRNA detection probe (Exiqon, Denmark) was used to detect the endogenous expression of miR-200b-3p. Deparaffinized lung sections were treated with 15 μg/mL Proteinase-K for 10 minutes at 37°C. The sections were hybridized at 50°C (30°C lower than the probe Tm) for 18 hours in hybridization mix containing 40 nM of miR-200b DIG-labeled probes or 40 nM of scrambled miRNA probes or positive control miRNA probes (miR-126). Sections were washed with decreasing standard saline citrate buffer concentration at hybridization temperature. For immunostaining, sections were incubated with 1:800 anti-DIG reagents (Roche) for 1 hour at room temperature followed by incubation with AP substrate (Roche) for 1.5 hours at 30°C. Nuclear Fast Red (Vector Laboratories) was applied and nuclei were counterstained. Sections were mounted using Eukitt (Sigma) and visualized under phase contrast microscope.
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