Ab76655

Manufactured by Abcam

Sourced in United States

Ab76655 is a laboratory equipment product. It is a device used for scientific research and analysis purposes. The core function of this product is to enable specific tasks or procedures within a laboratory setting. No further details can be provided while maintaining an unbiased and factual approach.

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3 protocols using ab76655

Notch Signaling Pathway Protein Detection

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Samples were run by SDS-PAGE and transferred to low autofluorescence polyvinylidene difluoride membrane (Immobilon, FL, Millipore). Primary antibodies were used at the concentration described below overnight at 4 °C in blocking solution (1% milk powder in PBS). Secondary antibody (IR-Dye conjugation goat anti-mouse or -rabbit LI-COR Biosciences) was used at 1:50,000 dilution in blocking solution for 1 h. The membrane was scanned on the LI-COR Odyssey (LI-COR Biosciences) using the Image Studio software.
Dilution of primary antibodies. 1:250: Notch1 (ab52627, Abcam), NICD (ab8925, Abcam), Hes1(ab71559, Abcam), Dll1 (ab76655, Abcam), ATG7 (ab52472, Abcam), Numb (ab14140, Abcam)
1:1,000: LC3 (NB 100-2220, Novus), ATG16L1 (pAb PM040, MBL), Beclin (#3738S, Cell Signalling), VAMP3 (gift from A.A. Peden)
1:2,000: Actin (A2066,Sigma-Aldrich).
Notch1 on western blot shows Notch NTMD (125 kDa). It is the form which is cleaved during maturation at the plasma membrane. The NICD (activated Notch1) antibody detects VLLSRKRRRQHGQC, a sequence, which is not accessible in the uncleaved form. It is exposed after S1 cleavage. The protein is detected at 80 kDa.

Wu X., Fleming A., Ricketts T., Pavel M., Virgin H., Menzies F.M, & Rubinsztein D.C. (2016). Autophagy regulates Notch degradation and modulates stem cell development and neurogenesis. Nature Communications, 7, 10533.

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Immunocytochemistry for Autophagy and Notch Signaling

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For immunocytochemistry, the cells were incubated in 200 nM Rapamycin (rap), 400 nM Bafilomycin A1 (baf), 100 mM Trehalose (treh) for 8 h or starved with HBSS for 2 h (1 × wash with HBSS before incubation for 2 h).
The cells were fixed for 3 min with ice cold methanol or for 10 min with 4% paraformaldehyde (PFA). Concentration of the primary and secondary antibodies are described below. The mounting solution was from Molecular Probes.
Dilution of primary antibodies. 1:50: Notch1 ms (ab44986, Abcam)
1:100: Notch1 rb (ab52627, Abcam), NICD (ab8925, Abcam), Hes1 (ab71559, Abcam), Pax6 (ab5790, Abcam), EEA1 (ab70521, Abcam), Dll1(ab76655, Abcam), ATG16L1 (Cell Signalling)
1:200: ATG9 (ab108338, Abcam)
1:300: LC3 (clone 5F10, Nanotools)
1:500: Tbr1 (ab31940, Abcam), Tbr2 (ab23345, Abcam), Nestin (ab6142. Abcam), 3β-tubulin (ab7751, Abcam)
1:800: AP2 (ab52222, Abcam)
The secondary antibodies Alexa 488, 568, 594 or 647 goat anti-mouse or goat anti-rabbit were obtained from Molecular Probes and used at 1:500.
Imaging was conducted with LSM710 Zeiss confocal with 63 × oil-immersion lense. The colocalization was measured using Volocity software for Mander's coefficient.

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Immunohistochemical analysis of Notch pathway in spinal cord injury

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Injured spinal cords of six rats from each group were removed at 1, 7, 14, 28 days and fixed in paraformaldehyde after cardiac perfusion. Serial sections (4 μm) were cut from a paraffin block. The sections were dewaxed and hydrated, and then were incubated for 2.5 min with 0.01 mol/L of citrate buffer for antigen thermal remediation. Endogenous peroxidase activity was quenched with a 10 min incubation in 3% H 2 O 2 at 37 C. These sections were blocked in 2% BSA for 10 min at 37 C and then were incubated with primary antibody for 16 hr at 4 C. The primary antibodies were anti-DLL1 antibody (ab76655) (1:1,600; Abcam, USA), anti-Presenilin1 (EP2000Y) antibody (ab76083) (1:100; Abcam), anti-Hes1 (EPR4226) antibody (ab108937) (1:300, Abcam) and anti-Hes5 antibody (ab107593) (1:400, Abcam). The sections were incubated with secondary antibody for 40 min at 37 C after washing with phosphate buffered saline (PBS). The sections were washed with PBS and stained with diaminobenzidine (DAB) solution for 5 min. The slices were observed under a light microscope, BX53 (Olympus Corporation, Japan). Microscopic pictures of the spinal cord tissue were captured at Â400 magnification, and Image-Pro plus 6.0 software was used to calculate the integral optical density (IOD) values of six images from each group.

Wang X., Wang Q., Tian H., Lv W., Song L., Li Z., Yao H, & Shi S. (2021). Electroacupuncture in promoting neural repair after spinal cord injury: Inhibiting the Notch signaling pathway and regulating downstream proteins expression. Anatomical record (Hoboken, N.J. : 2007), 304(11).

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