Cryosections were processed for in situ hybridization using previously established protocols (52 (link)). Briefly, Digoxigenin-labeled mRNA in situ probes diluted in hybridization buffer were added to the slides and incubated at 65°C overnight, followed by washes of high Stringency buffers and maleic acid buffer with tween (MABT) before blocking with 10% goat serum in MABT. Anti-Digoxigenin antibody diluted in MABT (with 1% goat serum) was added to the slides at 4°C overnight. Slides were then washed in MABT, followed by alkaline phosphatase buffer (NTMT). BM Purple (Millipore Sigma) was added, and the color reaction was allowed to develop overnight. Slides were then briefly washed in PBS and coverslipped with mounting medium for imaging. The probes used are listed in table S3.
Bm purple
Manufactured by Merck Group
Sourced in United States
BM Purple is a chromogenic substrate used for the detection of alkaline phosphatase activity. It is a sensitive and specific reagent that produces a purple-colored product upon enzymatic cleavage.
Automatically generated - may contain errors
Lab products found in correlation
T7 high yield rna synthesis kit, by New England Biolabs (4 mentions)
Anti dig fab fragment, by Roche (4 mentions)
Dig dutp, by Merck Group (3 mentions)
Proteinase k, by Promega (2 mentions)
Anti digoxygenin antibody, by Merck Group (2 mentions)
Axio zoom v16, by Zeiss (2 mentions)
W1 spinning disk confocal, by Nikon (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Entellan, by Electron Microscopy Sciences (1 mentions)
Anti digoxigenin ap fab fragments, by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Dmil microscope, by Canon (1 mentions)
Ds126311, by Canon (1 mentions)
Anti digoxigenin ap, by Merck Group (1 mentions)
Vector blue alkaline phosphatase substrate kit, by Vector Laboratories (1 mentions)
Dfc450 c camera, by Leica camera (1 mentions)
M250c leica stereomicroscope, by Leica camera (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
27 protocols using bm purple
1
In Situ Hybridization Protocol for Cryosectioned Samples
2
In-situ Hybridization of Cryo-Sections
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Cryo-sections were processed for in-situ hybridization using previously established protocols (52) . Briefly, DIG-labeled mRNA in situ probes diluted in hybridization buffer was added to the slides and incubated at 65C overnight, followed by washes of high stringent buffers and MABT before blocking with 10% Goat Serum in MABT. Anti-DIG antibody diluted in MABT (with 1% Goat Serum) was added to the slides for 4C overnight. Slides were then washed in MABT followed by Alkaline Phosphatase buffer (NTMT). BM Purple (Millipore Sigma) was added and the color reaction was allowed to develop overnight. Slides were then briefly washed in PBS and cover-slipped with mounting medium for imaging. The probes used are listed in Table 3.
3
Cell Surface Binding Assay for BMPR1A-LGR4
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Cell surface–binding assays were executed as previously described (14 (link)). In brief, HEK293T cells were transfected with BMPR1A-HA and LGR4 DNA and incubated with conditioned media in combination with 50 μM monomeric peptides or 20 μM dendrimers for 3 h or 16 h. Surface binding was detected by development with BM-Purple (catalog no.: 11442074001; Sigma). Images were obtained with DMIL microscope/Canon DS126311 camera (LEICA).
4
Colorimetric and Fluorescent In Situ Hybridization Protocols for Embryonic Tissue
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For colorimetric in situ hybridization embryos were treated with 10μg/mL proteinase K (Invitrogen 25530015) for 30 minutes, post-fixed, pre-hybridized at 68°C for 1hour and hybridized with 500ng/mL digoxigenin-labeled riboprobes at 68°C overnight as previously described (Lewandowski et al., 2015 (link)). BM purple (Sigma-Aldrich 11442074001) staining was visualized in dissected forelimbs using a table-top Leica light microscope equipped with a DFC420C Leica camera.
Fluorescent in situ hybridization was conducted using HCR v3.0 reagents as previously described (Ramachandran et al 2022). Briefly embryos were fixed overnight at 4°C in 4% PFA/PBS and rehydrates with MeOH/PBST (0.1% Tween-20). Embryos were then treated with 10μg/mL proteinase K for 30 minutes, incubated in 4nM probe overnight at 37°C, and then in 60pmol hairpin overnight at room temperature. After hairpin incubation, the samples were washed and counterstained in DAPI (1:5000 dilution; Life Technologies D1306), embedded in low-melt agarose and cleared in CeD3++ as described (Anderson et al., 2020 (link)). Samples were imaged on a Nikon W1 spinning disk confocal.
Fluorescent in situ hybridization was conducted using HCR v3.0 reagents as previously described (Ramachandran et al 2022). Briefly embryos were fixed overnight at 4°C in 4% PFA/PBS and rehydrates with MeOH/PBST (0.1% Tween-20). Embryos were then treated with 10μg/mL proteinase K for 30 minutes, incubated in 4nM probe overnight at 37°C, and then in 60pmol hairpin overnight at room temperature. After hairpin incubation, the samples were washed and counterstained in DAPI (1:5000 dilution; Life Technologies D1306), embedded in low-melt agarose and cleared in CeD3++ as described (Anderson et al., 2020 (link)). Samples were imaged on a Nikon W1 spinning disk confocal.
5
Receptor-RSPO Binding Assay in HEK293T Cells
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HEK293T cells were seeded in 24 well plates coated with Poly-D-Lysine (Sigma P6407). 250 ng ml−1 of human FGFR4-HA (Sino Biological HG10538-CY), BMPR1A-HA, Xenopus laevis Fgfr4a-V5, and Xenopus tropicalis Lgr4-V5 DNA were transfected in HEK293T cells and incubated with 2-2.5 U ml−1 AP-fused conditioned media for 3 h on ice. For competitive binding assays, cells were treated with RSPO2-AP in combination with either 5 nM human FGFR4 Fc Chimera (R&D systems 685-FR) or 5 nM BMPR1A Fc Chimera (Abcam ab238293). For peptide competition assays, cells were treated with RSPO2-AP in combination with 50 μM TK, KC and RW peptides. Receptors-RSPO binding was crosslinked with dithiobis (succinimidyl) propionate (DSP) (Thermo 22585) for 15 min on ice and additional 30 min at room temperature. Cells were washed three to four times with DPBS and treated with 2 mM Levamisole for 15 min to inactivate endogenous AP activities and developed with BM-Purple (Sigma 11442074001). Cells were mounted with Fluoromount G (Invitrogen 00495802). Representative images were obtained using LEICA DMIL microscope/Canon DS126311 camera.
6
Whole-mount in situ hybridization of Cronos and ttna
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Whole-mount in situ hybridization with digoxigenin-labeled mRNA antisense probes to Cronos and ttna was performed as previously described (Thisse and Thisse, 2008 (link)). For Cronos, an 89nt digoxigenin-labeled riboprobe unique to this isoform was hybridized at 60°C. For ttna, a 453nt riboprobe corresponding to a constitutive region was hybridized at 68°C. In both cases embryos were developed in BM purple (11442074001, Sigma-Aldrich).
7
Whole-Mount In Situ Hybridization in Xenopus
8
Xenopus tropicalis katnal2 Expression
9
Mouse Embryo Pax1 Expression Analysis
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Mouse E12.5 embryos were fixed in 4% paraformaldehyde. A plasmid containing mouse Pax1 (GenScript; OMu21524) was used as template for DIG-labeled probes. Mouse whole-mount in situ hybridization was performed according to standard procedures64 (link). Briefly, embryos were rehydrated and treated with Proteinase K (Promega; V3021). Following re-fixation and prehybridization, embryos were hybridized with a DIG-labeled probe. The hybridized probe was immunodetected with anti-digoxigenin Fab fragments conjugated to alkaline phosphatase (Sigma-Aldrich; 11093274910) and visualized with a Bmpurple (Sigma-Aldrich; 11442074001) according to the manufacturer’s protocol.
10
Whole-mount in situ hybridization of Esrp in Branchiostoma
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Adult Branchiostoma lanceolatum animals were collected in Banyuls (France) and Mataró (Catalonia). Spawning of reproductive individuals was done as previously reported64 (link). Embryos were cultured in filtered sea water at 17 °C and fixed in 4% PFA in MOPS buffer overnight at 4 °C. WMISH was performed with digoxigenin labeled RNA probes for Esrp65 (link). Hybridization temperature was 65 °C, and antibodies were incubated for 3–4 h, followed by overnight washes in MABT buffer (100 mM maleic acid, 150 mM NaCl, 0.1% Tween-20, pH 8) to reduce background. Detection was done with alkaline phosphatase-conjugated anti-digoxigenin (DIG) antibody. BM Purple (Sigma) was used as chromogenic substrate. A minimum of 15 embryos of the same stage was used to evaluate expression patterns. Sequences of oligonucleotides used in this section are found in Supplementary Table 2 .
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