In situ hybridization was carried out according to Yamamoto et al. (2004) (link) for gammaM2 crystallin or according to Ma et al. (2014) (link) for all other genes. The RNA probes used for in situ hybridization were prepared using the oligonucleotide primers shown in Table S1 . After hybridization the specimens were incubated in BM Purple AP Substrate (Roche) at room temperature in the dark. Whole mount stained specimens were cleared through an increasing glycerol series in PBS, then imaged by light microscopy and photographed. Some of the stained specimens were embedded in Paraplast, and sectioned at 10 μm. The sections were mounted on glass slides, imaged by light microscopy, and photographed.
Bm purple ap substrate
Manufactured by Roche
Sourced in Switzerland, United States
BM purple AP substrate is a colorimetric substrate used for the detection and visualization of alkaline phosphatase (AP) activity in various analytical applications. It provides a purple-colored reaction product upon enzymatic cleavage by AP.
Automatically generated - may contain errors
Lab products found in correlation
Dig rna labeling kit, by Roche (4 mentions)
Mircury lna probe, by Qiagen (3 mentions)
Pgem t easy vector, by Promega (3 mentions)
Dig rna labeling kit sp6 t7, by Roche (3 mentions)
Blocking reagent, by Roche (3 mentions)
Anti dig alkaline phosphatase antibody, by Roche (2 mentions)
T7 polymerase, by Roche (2 mentions)
Boehringer blocking reagent, by Roche (2 mentions)
Proteinase k, by Qiagen (2 mentions)
Axiocam ccd camera, by Zeiss (2 mentions)
Anti digoxigenin ap fab fragment, by Roche (2 mentions)
Axio imager, by Zeiss (2 mentions)
Smz1500 microscope, by Nikon (1 mentions)
Szx18, by Olympus (1 mentions)
Ds ri2, by Nikon (1 mentions)
Nuclear fast red, by Vector Laboratories (1 mentions)
Aquamount, by Merck Group (1 mentions)
Alkaline phosphatase conjugated anti dig fab fragments, by Roche (1 mentions)
Smz18 binocular, by Nikon (1 mentions)
Dig labeling mix, by Roche (1 mentions)
47 protocols using bm purple ap substrate
1
In situ Hybridization of Embryonic Genes
2
Whole-Mount In Situ Hybridization of Zebrafish
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Tissue-specific changes in the expression of selected genes of interest in the HPT axis were examined via WISH assays. Selected genes included thyroid receptors (thraa and thrb), deiodinases (dio1, dio2 and dio3b) and transthyretin (ttr). The WISH protocol used in this study was modified from Thisse and Thisse (Thisse and Thisse, 2008) and detailed methodologies are provided in Parsons et al., 2019 (Parsons et al., 2019) . Briefly, digoxigenin (DIG) antisense RNA probes were synthesised using purified zebrafish target gene DNA, that were subsequently treated with DNAse, purified by lithium chloride precipitation and diluted with hybridisation buffer. For WISH assays, fixed embryos were rehydrated through a series of PBS washes, treated with proteinase K and hybridised with an antisense probe (100 μL of hybridisation buffer containing approximately 15 ng of antisense DIG-labelled RNA probe) overnight at 65˚C. Embryos were subsequently incubated in blocking solution and then incubated with an anti-DIG antibody conjugated with alkaline phosphatase (Roche; x5000, diluted 1/100 with blocking solution). Embryos were then stained in BM-Purple AP Substrate (Roche) until signal or background staining became visible. Staining times varied depending on the probe (Table S2 andS3). Embryos were observed and photographed using Nikon SMZ1500 microscope equipped with a digital camera.
3
Visualizing BnaA3.NIP5;1 Expression in Rapeseed Roots
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Rapeseed root samples from 5-d-old seedlings grown under B-deficiency conditions were fixed with a solution consisting of 63% (v/v) ethanol, 5% (v/v) acetic acid and 2% (v/v) formaldehyde for 4 h, embedded into 5% (w/v) agarose and then sectioned to 50 μm. BnaA3.NIP5;1 in situ RT-PCR flowed method with the modifications of Athman et al. (2014) [46 (link)]. The samples were stained using BM purple AP substrate (Roche) for 30 min, washed in an orderly manner with washing buffer, mounted in 40% (v/v) glycerol and then observed under a microscope (Nikon DS-Ri 2).
The pQ::BnaA3.NIP5;1 fragment was amplified from the pQ::BnaA3.NIP5;1-GFP vector and cloned into pBI121 to generate pQ::BnaA3.NIP5;1-GUS constructs. The resulting vectors were transformed into W10 rapeseed. The pQ::BnaA3.NIP5;1-GUS transgenic seedlings were incubated in a solution of 1 mg ml-1 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc), 100 mM sodium phosphate (pH 7.0), 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 10 mM Na2EDTA, 0.1% (v/v) Triton X-100 and 20% (v/v) methanol at 37°C in the dark for 1 h. After incubation, the chlorophyll was removed using 75% ethanol, and images were taken using stereomicroscope (Olympus SZX18).
The pQ::BnaA3.NIP5;1 fragment was amplified from the pQ::BnaA3.NIP5;1-GFP vector and cloned into pBI121 to generate pQ::BnaA3.NIP5;1-GUS constructs. The resulting vectors were transformed into W10 rapeseed. The pQ::BnaA3.NIP5;1-GUS transgenic seedlings were incubated in a solution of 1 mg ml-1 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc), 100 mM sodium phosphate (pH 7.0), 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 10 mM Na2EDTA, 0.1% (v/v) Triton X-100 and 20% (v/v) methanol at 37°C in the dark for 1 h. After incubation, the chlorophyll was removed using 75% ethanol, and images were taken using stereomicroscope (Olympus SZX18).
4
In Situ Hybridization of Vertebrate Embryos
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Whole-mount and section in situ hybridization of lamprey, hagfish, and catshark embryos was performed as previously described [10 (link), 41 (link)]. In brief, paraffin-wax-embedded sections of embryos were deparaffinized, treated with proteinase K, and hybridized with DIG-labeled RNA probe overnight. Hybridized probes were detected with anti-DIG-AP Fab fragments and visualized with reaction with BM purple AP substrate (11442074001; Roche). Sections were counterstained with Nuclear Fast Red (H-2403; Vector Laboratories). The probe sequence of LjLbx-A is highlighted in Additional file 1 : Fig. S2. The probe template for EbLbx-A was amplified with the primers 5′-CGCCCTGGAGGAACTCGCGAG-3′ and 5′-CATTGCCCTTAGCGTAAAGCF-3′. For the catshark, to distinguish the expression of Lbx1 and Lbx2, probes were designed to include the regions highly diverged between the two genes. Primers used to amplify the probe templates were Lbx1, 5′-GGACCTGGAGGAGATGAAGG-3′ and 5′-GACATGCGATGCAACAAGG-3′; Lbx2, 5′-CGAAGGGACTATTCCTTCTCT–3′ and 5′-AGTCCCACCCCTTCCCCAACAA-3′.
5
In Situ Hybridization of miRNA in Tumor Tissues
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In situ hybridization was performed on formalin-fixed, paraffin-embedded sections (4 μm thickness) of tumor specimens. After processing with 3% H2O2, sections were treated with proteinase K (2 μg/ml) at 37 °C for 30 min, washed, and prehybridized for 2 h at 37 °C. Hybridization with digoxygenin (DIG)-labeled miRCURY LNA probes (probe sense: 5′-cACTTATCAGGTTGTATTATAa -3′; Biosense Bioscience Co., Ltd, Guangzhou, China) 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 1 h at room temperature. Staining was visualized by adding BM purple AP substrate (Roche, Basel, Switzerland) according to the manufacturer’s instructions.
6
Cloning and In Situ Hybridization of Hbp1 Fragments
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A 903 bp Fl-Hbp1 and a 573 bp ΔHbp1 3’UTR fragment were cloned from 12.5 dpc testis. Primers were Fl-Hbp1-F and Fl-Hbp1-R and ΔHbp1-F and ΔHbp1-R, listed in S1 Table . The amplified fragments were cloned into the pGEM-T-Easy vector (Promega, Madison, USA) and verified by sequencing. The sense and anti-sense probes were synthesised using T7 and SP6 RNA polymerases through in vitro transcription. Gonads/mesonephroi were dissected and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for several hours at 4°C. Whole mount in situ hybridization (ISH) with digoxygenin (DIG) labelled RNA probes was carried out as described by [25 (link)]. The RNA probe was detected by incubation with BM Purple, AP Substrate (Roche, Indianapolis, USA).
7
In Situ Hybridization of miR-207 in Cochlea
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ISH was performed to confirm the expression of miR-207 in cochlea hair cells in vivo. Cochleas of C57BL/6 mice were carefully and quickly resected, followed by removing the stapes from the oval window and opening the cochlea apex. We then placed the cochleas in fresh 4% paraformaldehyde (PFA) in PBS overnight at 4 °C. After decalcification with 0.1 M ethylenediaminetetraacetic for 3 days at 4 °C, the cochleas were embedded in paraffin and sectioned at 5 μm. ISH was performed as described.32 (link) Briefly, sections were hybridized with labeled LNA probe, which was detected using AP-conjugated sheep anti-DIG Fab fragment and BM Purple AP Substrate (Roche). The sections were then fixed by 4% PFA for 20 min finish with milliQ water (Millipore, Billerica, MA, USA) rinses. Slides were coverslipped with Aquamount (Merck, Darmstadt, Germany). The slides were observed using a light microscope.
8
Whole-mount in situ Hybridization of Mouse Embryos
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Whole-mount in situ hybridization was performed as described previously21 (link). Embryos were dissected at E9.5 and fixed with 4% paraformaldehyde in PBS for durations of 1 h to overnight. After dehydration using a methanol series, embryos were rehydrated with 0.1% Tween 20 in PBS (PBST), bleached with 6% H2O2 in PBS, treated with Protease K, and then hybridized with digoxygenin (DIG)-labeled probes at 68ºC overnight. Embryos were then washed with 5 × and 2 × SSC/50% formamide and PBST. To detect DIG-labelled probes, embryos were incubated with anti-DIG antibodies after treatment with blocking reagent for 1 h, and the signal was developed with BM purple AP substrate (Roche, Basel, Switzerland).
9
In situ Hybridization of Nasopharyngitis and NPC
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In situ hybridization was performed on formalin-fixed, paraffin-embedded sections (5 μm) of nasopharyngitis and NPC tissue. Briefly, after dewaxing, sections were treated with proteinase K (2 μg/ml) at 37 °C for 15 min, washed and prehybridized for 1 h at 49 °C. Hybridization with digoxigenin (DIG)-labeled miRCURY LNA probes (Exiqon) was performed overnight at 49 °C. Slides were then washed at 49 °C and incubated with alkaline phosphatase-conjugated anti-DIG Fab fragments (1 : 1500; Roche Applied Science, Indianapolis, IN, USA) for 1 h at room temperature. The staining was visualized by adding BM purple AP substrate (Roche Applied Science) according to the manufacturer's instructions.
10
Whole-mount in situ Hybridization Protocol for P2X Receptor Expression
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Whole-mount in situ hybridizations were carried out as previously described (Massé et al., 2006 (link), 2010 (link); Blanchard and Massé, 2019 (link)). In order to detect the expression of each p2rx subunit, fragments of their cDNA, except for p2rx4, were subcloned into plasmid pBSKS, as described in Supplementary Table S3 , in order to generate specific antisense and sense probes. Riboprobes were generated by in vitro transcription using a DIG RNA labeling kit following manufacturer’s recommendations (Roche) after linearization of the corresponding plasmid (see Supplementary Table S3 for details). Templates were designed in order to generate 250–600 bases riboprobes; when using larger riboprobes, a limited alkaline hydrolysis was performed. Riboprobe hybridization detection was carried out with an anti-DIG Alkaline Phosphatase antibody (Roche, Reference 11093274910) and the BM-Purple AP substrate (Roche Reference, 11442074001). After bleaching, embryos were photographed using the SMZ18 binocular (Nikon).
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