The largest database of trusted experimental protocols

3 protocols using anti nse

1

Immunohistochemical Detection of CD31, NSE, HIF1, and HIF2

Check if the same lab product or an alternative is used in the 5 most similar protocols
For detection of CD31 antigen, slides were digested with protease I for 20 min, then incubated with anti-DC31 (DAKO, Carpinteria, CA, 1/20) for 32 min and run on Ventana BenchMark XT with Ultra View kit. For detection of NSE, slides were exposed to antigen retrieval buffer (citrate buffer ph 6.0) for 20 min prior to incubation with anti-NSE (DAKO, Carpinteria, CA, 1/2000).
A modified protocol was used for detection of expression of HIF1 and HIF2. For antigen retrieval, sections were immersed in preheated Dako target retrieval solution in a pressure cooker for 90 s. Primary antibodies were: mouse monoclonal anti-human HIF1α H1α67 (Novus Biologicals, Littleton, CO) 1/4000; rabbit polyclonal anti-mouse HIF2α PM8 antiserum (Novus Biologicals, Littleton, CO), 1/100. Primary antibody was omitted for negative controls. Antigen/antibody complexes were revealed by means of the Catalyzed Signal Amplification system (DAKO, Carpinteria, CA, Dako) (HIF) according to the manufacturer’s instructions.
+ Open protocol
+ Expand
2

Quantifying Protein Expression via Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols
Protein concentration was measured using the bicinchoninic acid (BCA) method (Thermo Scientific). Subsequently, 20 μg of cell lysate was denatured with sample buffer (62.5 mM Tris–HCl pH6.8, 2% (v/v) SDS, 50 mM DTT, 10% (v/v) glycerol), subjected to 4%–20% gradient SDS-PAGE, and transferred onto nitrocellulose membranes (BioRad). The membranes were blocked for 1 h with 5% (w/v) dried skimmed milk in TBS-T buffer (25 mM Tris, 150 mM NaCl, and 0.1% (v/v) Tween-20, pH 7.4), and incubated with the following primary antibodies: mouse anti-GFAP (Sigma-Aldrich; 1:50,000), rabbit anti-Iba1 (Wako; 1:10,000), mouse anti-β-actin (Sigma-Aldrich; 1:20,000), anti-NSE (1:1000, Dako), or anti-GAPDH (1:40000, GeneTex), at 4°C overnight. Then, membranes were incubated with the corresponding HRP-coupled secondary antibodies (Jackson Immunoresearch; 1:10,000) for 1 h at room temperature, and the bands were visualized using the Super Signal West Pico chemiluminescent substrate (Thermo Scientific). The immunoreactive signals were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Iba1 and GFAP values were normalized to β-ACTIN values. NSE results, due to molecular weight, were normalized to GAPDH values.
+ Open protocol
+ Expand
3

Lineage Tracing of CgA-Expressing Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
CgA-CreERT2 mice were crossed with Rosa-CAG-LSL-ZsGreen1 Cre-reporter mice. The offspring were intraperitoneally injected with tamoxifen (1 mg/body) dissolved in corn oil once daily for 3 days. The duodenum, pancreas, and brain were resected and fixed with 10% formalin. Paraffin-embedded tissue sections were immunostained with anti-chromogranin A, anti-insulin, and anti-NSE (DAKO, Glostrup, Denmark) as described [17 (link)]. The sections were visualized with fluorescently (Alexa 555) labeled secondary antibodies (Life Technologies) on a BZ9000 microscope (KEYENCE, Tokyo, Japan).
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!