Small intestines were collected from embryos at desired stages and fixed in 4% paraformaldehyde in PBS and embedded in OCT, allowing for 14μm transverse sections of the gut tube. CD44 immunohistochemistry was performed with rat anti-CD44 (v6) (1:100 Biosciences) and detected using the Anti-Rat HRP-DAB Cell & Tissue Staining Kit (R & D Systems). The following antibodies were used for immunofluorescence staining at the listed concentrations: Sox9 (1:100, R & D Systems), β-catenin (1:100, Sigma), PDGFα (1:100 in chick, 1:300 in mouse, Santa Cruz), FITC-conjugated Smooth muscle actin (1:100, Abcam), phospho-SMAD 1/5 (1:300, Cell Signaling), Shh (5E1, 1:20). Sections were incubated with primary antibody overnight at 4 degrees and then incubated with Alexa secondary antibodies used at 1:300 for 2 hours at room temperature. DAPI (molecular probes) was used as a nuclear counter stain. 100μM Edu (Invitrogen) was added to guts in culture and samples were harvested after 4 hours of Edu incubation. Edu was detected in sectioned tissue using the Click-iT Edu system (Invitrogen).
Click it edu system
Manufactured by Thermo Fisher Scientific
The Click-IT EdU system is a fluorescence-based reagent kit designed for the detection and quantification of DNA synthesis in proliferating cells. It provides a sensitive and specific method for labeling and visualizing newly synthesized DNA.
Automatically generated - may contain errors
Lab products found in correlation
C2 laser scanning head, by Nikon (2 mentions)
Alexa fluor 594, by Merck Group (2 mentions)
Cardiac troponin t, by Thermo Fisher Scientific (2 mentions)
Phospho smad1 5, by Cell Signaling Technology (1 mentions)
β catenin, by Merck Group (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
Cardiac α actinin, by Merck Group (1 mentions)
Eclipse ti confocal microscopy, by Nikon (1 mentions)
Runx1, by Cell Signaling Technology (1 mentions)
3 protocols using click it edu system
1
Embryonic Gut Development Analysis
2
Immunostaining and EdU Labeling in Mouse Hearts
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For immunostaining, hearts were fixed in 4% PFA at 4 °C overnight, embedded in OCT compound (Sakura), and sectioned at 8 μm thickness. For PCM1 staining, we used fresh-frozen (non-fixed) samples and sections were fixed in 10% formalin for 10 min. Sections were blocked with 1% bovine serum albumin (BSA), incubated with primary antibodies against PH3 (rabbit polyclonal, 1:200; Millipore), PCM1 (1:1000; Sigma), cardiac α-actinin (1:200; Sigma), cardiac Troponin T (1:200; Thermo), YAP (1:100; Cell Signaling), and smooth muscle α-actin conjugated with AlexaFluor594 (1:200; Sigma), and were further incubated with Alexa Fluor-conjugated secondary antibodies against mouse or rabbit IgG and with DAPI. For EdU staining, postnatal mice were administered an intraperitoneal (IP) injection of EdU (5 μg/g of mouse body weight) at P5, P6, P12, and P13, and we collected the hearts at P14. EdU incorporation was assessed using Click-IT EdU system (Invitrogen). Fluorescent images were captured using Eclipse Ti confocal microscopy with a C2 laser-scanning head (Nikon).
3
Multimodal Cardiac Tissue Analysis
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For immunostaining, hearts were fixed in 4% PFA at 4 ℃ overnight, embedded in OCT compound (StatLab), and sectioned at 8m thickness. For PCM1 staining, we used fresh-frozen (non-fixed) samples and sections were fixed in 10% formalin. Sections were blocked with 1% bovine serum albumin (BSA), incubated with primary antibodies against PH3 (rabbit polyclonal, 1:200; Millipore), PCM1 (1:1000; Sigma), cardiac -actinin (1:200; Sigma), cardiac Troponin T (1:200; Thermo), YAP (1:100; Cell Signaling), Runx1 (1:100; Cell Signaling), smooth muscle-actin conjugated with AlexaFluor594 (1:200; Sigma), and were further incubated with Alexa Fluorconjugated secondary antibodies against mouse, rabbit IgG and with DAPI. For EdU staining, neonates were administered an intraperitoneal (IP) injection of 5-ethynyl-2-deoxyuridine (EdU, 5g/g of mouse body weight). EdU incorporation was assessed using Click-IT EdU system (Invitrogen). Fluorescence images were captured using Eclipse Ti confocal microscopy with a C2 laser-scanning head (Nikon).
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