Sagittal serial sections were stained utilizing primary antibodies directed to mouse Sost (R&D Systems, Minneapolis, MN, USA; AF1589 [1 µg/mL]), human SOST (Abcam, Cambridge, MA, USA; ab75914 [100 µg/mL]), Collagen II (Abcam; ab21291 [1:50]), activated β‐catenin (Millipore, Billerica, MA, USA; 05‐665 [10 µg/mL]), MMP 2 (Abcam; ab110186 [5 µg/mL]), MMP 3 (Abcam; ab52915 [7.04 µg/mL]), MMP9 (Abcam; ab137867 [10.22 µg/mL]), MMP14 (Abcam; ab53712 [10 µg/mL]), and Furin (Abcam; ab3467 [20 µg/mL]). Trypsin/EDTA was used for antigen retrieval in 37°C for 30 minutes for all primary antibody except for activated β‐catenin and MMPs 2, 3, 9, and Furin, which required Uni‐trieve (Innovex Biosciences, Richmond, CA, USA) in 65°C for 30 minutes. After Uni‐trieve, activated β‐catenin and Furin require an additional retrieval with Proteinase K (Ambion, Austin, TX, USA; AM2546 [20 µg/mL]) for 20 minutes. This was followed by using Alexa‐Fluor 488 (green) or 594 (red) (Molecular Probes, Eugene, OR, USA) to determine protein expression.
Activated β catenin
Manufactured by Merck Group
Sourced in United States
Activated β‐catenin is a lab equipment product used for research purposes. It is a protein involved in cellular signaling pathways. The core function of this product is to enable the study and analysis of β‐catenin activation, which is crucial for various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Ab75914, by Abcam (1 mentions)
Ab52915, by Abcam (1 mentions)
Ab137867, by Abcam (1 mentions)
Ab53712, by Abcam (1 mentions)
Ab110186, by Abcam (1 mentions)
Ab3467, by Abcam (1 mentions)
Af1589, by Abcam (1 mentions)
Alexa fluor 488 green, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Total β catenin, by Cell Signaling Technology (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Cyclin d1, by Thermo Fisher Scientific (1 mentions)
β catenin, by Thermo Fisher Scientific (1 mentions)
Phosphorylated stat3, by Cell Signaling Technology (1 mentions)
Phosphorylated akt ser, by Cell Signaling Technology (1 mentions)
Phosphorylated gsk3β, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
7 protocols using activated β catenin
1
Immunohistochemical Analysis of Bone Markers
2
Western Blot Analysis of β-Catenin
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Protein lysates were obtained from isolated GFP+ cells or cultured CFs. Standard western blots were performed. Briefly, 15–30 μg of protein was separated by 10–15% SDS-PAGE gel and transferred to PVDF membranes. After blocking in Odyssey blocking buffer (LI-COR, P/N 927-40000), blots were probed with primary antibodies: activated β-catenin (1:500, Millipore, #05-665), total β-catenin (1:1000, Cell signaling, #9581 S), and GAPDH (1:10 000; Santa Cruz Biotechnology #25778). After incubation with corresponding anti-mouse/rabbit secondary antibodies (1:10 000; LI-COR), immunoblots were developed using ODYSSEY Infrared Imaging System (LI-COR). Signal intensities were quantified with ImageStudio software (LI-COR). GAPDH was used as a loading control. Uncropped images of western blots are included in Supplementary Fig. 9 .
3
Quantitative Western Blotting Assay
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Western blotting was performed as previously described (Merino et al., 2016 ) using antibodies to phosphorylated AKT‐Ser (#4058S), phosphorylated GSK3‐β (#9323P), and phosphorylated STAT3 (#9131) from Cell Signaling, Danvers, MA, USA; activated β‐catenin (#05665), STAT3 (#06596), and GAPDH (#MAB374) from Millipore, Billerica, MA, USA; β‐catenin (#138400) and cyclin D1 (#AHF0102) from ThermoFisher Scientific.
4
Immunohistochemical Analysis of Cell Markers
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Immunohistochemical staining was performed according to a previously established protocol28 (link). Primary antibodies against ACTA2 (Sigma-Aldrich, St. Louis, MO, USA), VIM (Santa Cruz Biotechnology, California, USA), FSP-1 (Thermo Scientific, Fremont, CA, USA), PCNA (Dako, Glostrup, Denmark), activated β-catenin (Sigma-Aldrich), and TAZ (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. TUNEL staining was performed using a commercial kit (Millipore, Billerica, MA, USA), in accordance with the manufacturer’s instructions.
5
Western Blot Analysis of Protein Targets
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The total proteins isolated from cells or tissue samples and protein concentration were measured by DC Protein Assay (Bio-Rad Laboratories). 10 µg of proteins from each sample were separated on 10% SDS–PAGE and then transferred onto PVDF membranes. After blocking with 5% bovine serum albumin, blots were incubated with primary antibodies overnight at 4 °C. The primary antibodies used are as follows: activated β-catenin (05-665, Sigma), Cyclin D1 (#2922 S, Cell Signaling Technology), β-Actin (#4970, Cell Signaling Technology). Membranes were then incubated with secondary antibodies for 1 h at room temperature. Membranes were exposed to Pierce ECL Western Blotting Substrate (GE Healthcare). Band intensities were determined using ImageJ (National Institutes of Health).
6
Immunohistochemistry of Brain Sections
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Brain sections were incubated with primary antibodies overnight, followed by appropriate fluorescently conjugated secondary antibodies and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542). Primary antibodies used were against BrdU (1:500; Abcam, ab6326), GFP (1:1000; Aves Lab, GFP-1020), Ki67 (1:300; Abcam, ab15580), phosphohistone H3 (1:1000; Upstate, 06-570), Tbr1 (1:500; Abcam, ab31940), Tbr2 (1:500; Millipore, ab31940), Par3 (1:500; Millipore, MABF28), Pax6 (1:500; Millipore, AB2237), anti-NeuN (1:1000; Millipore, ABN78), Cux1 (1:500; Santa Cruz Biotechnology), and activated β-catenin (1:500; Merck, 05-665). Secondary antibodies were from Vector Lab [biotinylated anti-rat immunoglobulin G (IgG; 1:200; BA-9400) and biotinylated anti-rabbit IgG (1:200; BA-1000)], from Jackson ImmunoResearch [Cy3 Fab fragment anti-rabbit IgG (1:200; 711-167-003), Alexa Fluor 488 anti-chicken IgY (1:200; 703-545-155), and Cy5 streptavidin (1:200; 016-170-084)], and from Invitrogen [Alexa Fluor 555 anti-rabbit IgG (1:200; A-31572)].
7
Immunofluorescence Staining of Intestinal Crypts
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Immunofluorescence staining was conducted as described by Mahe et al. (23 (link)). After collection medium was removed, crypts were fixed with 4% paraformaldehyde and permeabilized with Triton X-100 (0.1% in PBS). They were washed with PBS before fixation and again before permeabilization. After permeabilization, crypts were blocked with blocking buffer (1% BSA, 3% normal goat serum, and 0.2% Triton X-100 in PBS) and incubated with antibodies for BMI1 (Miltenyi), β-catenin (Miltenyi), activated β-catenin (EMD Millipore), CD24 (BioLegend), CD44 (Tonbo), CD66a (eBioscience), LGR5 (Miltenyi and Origene), or LRIG1 (R&D) (Table 2 ) in working buffer (0.1% BSA, 0.3% normal goat serum, and 0.2% Triton X-100 in PBS) before mounting (Fluoromount-G, SouthernBiotech) for observation. Crypts were washed with PBS before blocking, incubation, and mounting. Justification for specific antibodies used is provided in the Supplementary Methods (see Supplementary Digital Content 1, http://links.lww.com/CTG/A612 ). Note that 2 separate LGR5 antibodies were tested for immunofluorescence.
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