Four-micron-thick brain coronal sections were prepared as described earlier, deparaffinized in xylene, rehydrated via alcohol gradients, and washed with PBS (0.01 M, pH 7.4). Following antigen retrieval, the sections were blocked in 5% bovine serum albumin for 30 min at room temperature (RT) and incubated with primary antibodies as follows: rabbit anti-activated Notch1 (1:200; Ab52301, Abcam, Cambridge, UK); goat anti-Iba1 (1:300; Ab5076, Abcam); mouse anti-CD68 (1:200; Ab31630, Abcam); rabbit anti-NeuN (1:200, 24307T, Cell Signaling Technology, Danvers MA, USA) overnight at 4 °C. The next day, the slices were washed with PBS and incubated with secondary antibodies: donkey anti-goat Alexa 488 (1:500; A11055, Invitrogen), donkey anti-rabbit Alexa 555 (1:500; A31572, Invitrogen), donkey anti-mouse Alexa 488 (1:500; A21202, Invitrogen), donkey anti-goat Alexa 555 (1:500; A21432, Invitrogen), goat anti-mouse Alexa 555 (1:500; A21422, Invitrogen), and donkey anti-rabbit Alexa 488 (1:500; A21206, Invitrogen) for 1 h at RT. After washing with PBS three times, the sections were stained by 4′6-diamidino-2-phenylindole for 10 min at room temperature and observed under a fluorescence microscope (Leica-DMI8, Leica Microsystems, Wetzlar, Germany).
Ab52301
Manufactured by Abcam
Sourced in United Kingdom
Ab52301 is a lab equipment product from Abcam. It is a protein-based reagent used for research purposes. The core function of this product is to detect and measure target proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (2 mentions)
Ab52627, by Abcam (2 mentions)
H3k27me3, by Merck Group (2 mentions)
Ecl reagent, by GE Healthcare (2 mentions)
β actin, by Merck Group (2 mentions)
Bcl 2, by BD (2 mentions)
A21422, by Thermo Fisher Scientific (1 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
A21432, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Ab5076, by Abcam (1 mentions)
Ab31630, by Abcam (1 mentions)
Ab280898, by Abcam (1 mentions)
Ab114133, by Abcam (1 mentions)
Alexa fluor 555 donkey anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Sc 2028, by Santa Cruz Biotechnology (1 mentions)
Donkey anti goat igg hrp sc 2020, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 633 donkey anti goat igg antibody, by Thermo Fisher Scientific (1 mentions)
12 protocols using ab52301
1
Immunohistochemical Analysis of Activated Notch1, Microglia, and Neurons
2
Notch1, Runx2, and NICD Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies for immunoblot in this study included the following: anti-Notch1 (ab280898; Abcam, Cambridge, UK), anti-Runx2 (ab114133; Abcam), anti-NICD (ab52301; Abcam), anti-GAPDH (ab8245; Abcam). Primers for qPCR in this study included the following: Notch1 forward 5′-GCGACAACGCCTACCTCTG-3′, Notch1 reverse 5′-AAGCCATTGATGCCGTCC-3′, Runx2 forward 5′-GGACGAGGCAAGAGTTTCAC-3′, Runx2 reverse 5′-GAGGCGGTCAGAGAACAAAC- 3′, NICD forward 5′-CTATAGACGACATTGACGAGTGTGAC-3′, NICD reverse 5′-TGTAGAATTCAGAGGACAGTTGCACTTG-3′, GAPDH forward 5′-AATCCCATCACCATCTTCCAG- 3′, GAPDH reverse 5′-GAGCCCCAGCCTTCTCCAT-3′.
3
Antibody Characterization for Notch1 and NeuN Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Notch1 antibody (ab27526), Chac1 antibody (ab76386), Rb pAb to activated Notch1 (ab52301), Rb mAb to NeuN (ab177487), Ms mAb to NeuN (ab104224) were from Abcam. β-tubulin (sc-9014), TGN 38 (sc-27680), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025) and normal goat IgG (sc-2028) were from Santa Cruz Biotechnology. Anti-Botch1/Chac1 (75-181) was from Neuromab. Cleaved caspase-3 (D175) was from Cell Signaling Technology. Protein A+G Agarose (P2012) was from Beyotime. Secondary antibodies for western blot analysis, including goat anti-rabbit IgG-HRP (sc-2004), donkey anti-goat IgG-HRP (sc-2020), and goat anti-mouse IgG-HRP(sc-2005) were from Santa Cruz Biotechnology. Secondary antibodies for immunofluorescence, including Alexa Fluor-488 donkey anti-rabbit IgG antibody (A21206), Alexa Fluor-488 donkey anti-mouse IgG antibody (A21202), Alexa Fluor-555 donkey anti-mouse IgG antibody (A31570), Alexa Fluor-555 donkey anti-rabbit IgG antibody (A31572), and Alexa Fluor-633 donkey anti-goat IgG antibody (A21082) were from life technologies.
4
Western Blot Analysis of Melanoma Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot assays were performed as previously described.17 (link) In brief, total proteins from melanoma cells were extracted in RIPA buffer (Sigma-Aldrich; Merck KGaA) on ice (the lysis buffer contain phosphatase and protease inhibitors), Then, the lysates were prepared with lysis buffer (Beyotime), and the protein concentration was determined via the BCA assay. Approximately 50ug of protein was loaded into a 12% SDS/Polyacrylamide gel. Electrophoresis was carried out at 80 V for 20 min followed by 40 min at 120 V. Western blot analysis was performed following protein transfer to a PVDF membrane. After blocking with 5% nonfat milk, the membranes were incubated at 4 °C overnight with SOX10 (ab227684, Abcam), Notch1 (ab52627, Abcam), NICD (ab52301, Abcam), Hes1 (ab108937, Abcam), and GAPDH (ab8245, Abcam) antibody. Membranes were visualised with Electrochemiluminescence Plus Detection Reagents.
5
Immunoblotting Protein Expression Analysis
6
Western Blotting Analysis of BMEC Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed western blotting as previously described39 (link). Cell lysates were prepared from BMECs transfected with agomiR-497∼195, antagomiR-497∼195, Fbxw7 siRNA, P4HTM siRNA or their negative controls in cell lyses buffer with 2% sodium dodecyl sulfate, 2 M urea, 10% glycerol, 10 Mm Tris-HCl (pH 6.8), 10 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. Total cell lysates were separated by SDS–polyacrylamide gel electrophoresis blotted on polyvinylidene difluoride membranes (Millipore). The membranes were incubated with specific antibodies to Fbxw7 (Abcam, ab109617, 1:1,000), P4HTM (Abcam, ab94626, 1:500), HIF-1α (Abcam, ab16066, 1:2,000) and NICD (Abcam, ab52301, 1:1,500) then reprobed with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, 1:10,000). Blots were developed using an ECL Kit (Santa Cruz), and exposed to x-ray films. The uncropped scans of blots are shown in Supplementary Figs 9 and 10 .
7
Immunoblotting Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting was performed using the following primary antibodies EZH2 (1:1000, 612667, BD Bioscience), H3K27me3 (1:1000, 07–449, Millipore), H3 (1:2000, 4499, Cell Signaling), NICD (1:1000, ab52301, Abcam), Bcl-2 (1:1000, 551107, BD Bioscience), and β-actin (1:5000, A5441, Sigma). Signals were detected by ECL reagent (GE Healthcare).
8
Comprehensive Protein Expression Analysis
9
NOTCH-1 Protein Expression Analysis in H1993 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 24 h post-transfection, H1993 cells were counted and total proteins in 4 × 105 cells were extracted using RIPA solution (GenePharma). Total proteins were denatured in boiling water for 5 min. Proteins were then separated by 10% SDS-PAGE gel, transferred to PVDF membranes, and PBS (5% non-fat milk) was used to block membranes for 1 h at 24 °C. After that, rabbit anti-NOTCH-1 (1: 1200, ab52301, Abcam) and GAPDH (1: 1200, ab37168, Abcam) primary antibodies were used to incubate the membranes for 18 h at 4 °C. After that, further incubation with IgG H&L (IgG) (1:1000; ab6721; Abcam) secondary antibody was performed for 2 h at 24 °C. Signal development was performed by incubating membranes with RapidStep™ ECL detection reagent (EMD Millipore) for 5 min. Signals were processed using Image J v1.47 software.
10
Western Blot Analysis of Myocardial Infarction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from the myocardium around the infarcted area (Solarbio, China), and the concentration was measured using a bicinchoninic acid protein concentration kit (Beyotime, China). The protein samples were separated by 10% acrylamide gel electrophoresis and transferred to nitrocellulose membranes, which were later incubated in the first antibody at 4°C overnight. The following target protein was detected to obtain their expression level: Jagged-1 (ab109536, Abcam, United Kingdom), Notch1 (3608, CST, United States), NICD (ab52301, Abcam, United Kingdom), Hes-1 (11988, CST, United States), GAPDH (Hangzhou Good Here Biotechnology, China). After the membranes were washed three times with PBS-T (PBS containing 0.5% Tween 20), they were further incubated in the secondary antibody (Jackson ImmunoResearch, United States) for 1 h at room temperature away from light. Then, bands were acquired using ECL visualization. The images were analyzed by the Image-J software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!