At the end of each treatment, C2C12 cells were washed twice with ice-PBS 1X and lysed in ice using Ripa Buffer (50 mM Hepes, 150 mM NaCl, 0,1% SDS, 1%TRITON 100x, 1% deoxycholate acid, 10% glycerol, 1,5 mM MgCl2, 1 mM EGTA, 1 mM NaF; all purchased from Sigma-Aldrich) supplemented with 2 mM sodium orthovanadate (Sigma-Aldrich), 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich) and 1:100 mix Protease Inhibitor Cocktail (Sigma-Aldrich). 40 μg of protein of each sample was resolved into 8% and 15% SDS-PAGE gels, and polyvinylidene difluoride (PVDF) membranes (GE Healthcare) were incubated overnight at 4 °C with specific primary antibody: anti-rabbit Cyclin D1 (1:1000, Cell Signalling), anti-mouse Desmin (1:1000, Santa-Cruz), anti-rabbit Phospho-AMPK (1:1000, Millipore), anti-mouse AMPK (1:500, Santa-Cruz), anti-mouse Phospho-JNK (1:500, Santa-Cruz), anti-mouse JNK (1:500, Santa-Cruz) and anti-mouse SMA (1:1000, Santa-Cruz). Protein expression was normalized and verified through anti-mouse β-actin detection (1:5000; Sigma-Aldrich) and reported as mean ± SD (% vs control).
Deoxycholate acid
Manufactured by Merck Group
Deoxycholate acid is a bile acid that is commonly used as a component in laboratory media and buffers. It serves as a surfactant, aiding in the solubilization and emulsification of substances in aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Sodium orthovanadate, by Merck Group (1 mentions)
Phenylmethanesulfonyl fluoride, by Merck Group (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Triton 100x, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Normal goat serum (ngs), by MP Biomedicals (1 mentions)
Mowiol mounting medium, by Merck Group (1 mentions)
Tcs sp5, by Nikon (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 protocols using deoxycholate acid
1
Western Blot Analysis of C2C12 Cell Signaling
2
X-Gal Staining of Frozen Retinal Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen retinal sections were fixed in cold X-gal fix (1% formaldehyde, 0.2% glutaraldehyde (Sigma–Aldrich)) for 10 min at 4 °C then washed 3 times for 10 min in X-gal buffer (5 mM EGTA (pH 8.0), 2 mM MgCl, 0.04% NP-40, 0.01% deoxycholate acid (Sigma–Aldrich) in PBS, pH 7.9), before staining with X-gal stain (5 mM potassium hexacyanoferrate (III) (K3Fe), 5 mM potassium ferricyanide (K4Fe), 0.5% X-gal substrate (Fisher Scientific) in X-gal buffer) at 37 °C overnight following an in-house optimized protocol. Briefly, X-gal buffer was prepared by adding 0.045 g (K3Fe), and 0.06 g (K4Fe) in 25 mL X-gal buffer. 250 μL X-gal substrate was added to the mix shortly before staining. Sections were incubated with X-gal stain at 37 °C in the dark overnight. Sections were washed 3 times in X-gal buffer and post-fixed in 4% PFA for 1 h at 4 °C. Sections were dehydrated through a graded ethanol series and cleared in a CLERENE Solvent (LEICA) twice for 5 min. Sections were immediately mounted in DPX mounting medium.
3
Brain Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence, brains were dissected and transferred to a tube containing 100 μl ice cold DPBS solution. After centrifugation at 800 xg for 5 min, the supernatant was replaced by 4% Formaldehyde in PBT 0.3% (DBPS + 0.3% Triton X-100 (Sigma)) and incubated at room temperature with rotation for 15 min. Brains were washed with PBT 0.3% three times, rotating for 10 min at room temperature each time and then blocked in Pax-DG (10 g BSA (Sigma), 3g Deoxycholate Acid (Sigma), 3ml Triton X-100 (Sigma), 50ml Normal Goat Serum (MP Biomedicals), 100ml 10X PBS, 850ml H2O) for 2 hours at room temperature with rotation. Primary antibody mixes were created in Pax-DG (dilutions detailed in the Key Resources Table) and brains were incubated in these mixes overnight at 4°C with rotation. The next day, brains were washed with PBT 0.3% three times, rotating for 10 min at room temperature each time and then stained with secondary antibody mixes in Pax-DG (dilutions detailed in the Key Resources Table) for 2 hours at room temperature with rotation. Brains were washed with PBT 0.3% three times, rotating for 10 min at room temperature each time and mounted in Mowiol mounting medium (Sigma). Imaging was performed using the Leica TCS SP5 and Nikon Spinning Disk confocal microscopes.
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!