Paraffinized clinical melanoma biopsy sections were provided by the Tissue Procurement Core Facility at the University of Iowa. MC1R expression in representative clinical melanoma samples from early stage T1 to late stage T4 (n = 6) was analyzed by immunohistochemical (IHC) staining. Briefly, samples were deparaffinized by three cycles of 100% xylene (3 min cycle−1), two cycles of 100% ethanol (3 min cycle−1), one cycle of 95% ethanol (1 min), and one cycle of 70% ethanol (1 min). Antigen retrieval was performed in 10 mM citrate buffer (pH = 6) at 95 °C for 15 min. The sections were blocked in 5% horse serum (Sigma, H1046) for 1 h, followed by incubation with rabbit monoclonal anti-MC1R (1:100 dilution, ab125031, Abcam) at 4 °C overnight. Secondary antibody incubation used HRP goat anti-rabbit IgG antibody-peroxidase (PI-1000–1, Vector Labs). The samples were finally stained with ImmPACT NovaRED Peroxidase (HRP) Substrate (SK-4805, Vector Labs). Bright-field microscopy was performed using an Olympus BX-61 instrument in the Central Microscopy Research Facility at the University of Iowa.
Ab125031
Manufactured by Abcam
Ab125031 is a recombinant monoclonal antibody that can be used for research purposes. This product is designed for in vitro experimental use only.
Automatically generated - may contain errors
2 protocols using ab125031
1
Immunohistochemical Analysis of MC1R in Clinical Melanoma
2
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lyzed in 6×SDS-PAGE sample buffer. The lysates of cells were loaded on a 12% polyacrylamide gel and separated under reducing conditions, using Rainbow-colored protein molecular weight markers as a reference. At the end of the electrophoresis, the gel was soaked in Western transfer solution before electrophoretic transfer of the protein onto polyvinylidene difluoride membrane at 120 v for 120 min. The membrane was blocked for 1 h in TBST containing 5% dried skimmed milk powder. After three washes in TBST, the membranes were incubated with primary antibody at a 1:1,000 dilution: Tyrosinase (ab170905; Abcam), Mc1R (ab125031; Abcam) and β-actin (12620; Cell Signaling Technology), which for 1 h at room temperature followed overnight at 4℃. After three washes with TBST, the membranes were then incubated with a 1:1,000 dilution of HRP-conjugated goat anti-rabbit IgG antibody for 1 h at room temperature and developed with the ECL Western blotting system according to the supplier's recommendations. The membranes were then exposed to X-ray film which was subsequently developed.
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!