The largest database of trusted experimental protocols

2 protocols using ch25h

1

Western Blotting of Cardiac Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
H9C2 cardiomyocytes were lysed in iced RIPA buffer (Solarbio, China) containing protease inhibitors for 30 min and centrifuged at 13523 g, 4°C for 10 min to collect the supernatants. The protein concentration was measured by the BCA protein assay kit (Solarbio). Proteins were then separated in sodium dodecyl sulfate (SDS) polyacrylamide gel by electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). Membranes were blocked with tris-buffered saline with tween 20 (TBST) buffer containing 5% nonfat milk and then incubated with primary antibodies against GAPDH (1:1000, Abcam, UK), Caspase-1 (1:1000, Abcam), NLRP3 (1:1000, Proteintech, China), GSDMD (1:1000, Biorbyt, UK), IL-1β (1:1000, Beyotime, China), and CH25H (1:500, Santa Cruz, USA) overnight at 4°C. After washing with TBST buffer three times, membranes were incubated with secondary antibody (Cell Signaling Technology, USA) for 2 h. The specific protein bands were detected using the Odyssey CLx imaging system (LI-COR, USA).
+ Open protocol
+ Expand
2

Immunohistochemical Analysis of Adipose Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols
Standard hematoxylin/eosin staining was performed on 5 μm paraffin-embedded tissue sections after deparaffinization. For UCP1 staining, 5 μm paraffin-embedded tissue sections were blocked with 2.5% normal goat serum in PBST (Phosphate-buffer saline with 0.1% Tween 20) for 1 h at room temperature following deparaffinization. UCP1 primary antibody (1:50, Sigma-Aldrich) was applied overnight at 4 °C. The following day, secondary antibody against rabbit (SignalStain Boost IHC, Cell Signaling) was applied for 1 h at room temperature and developed with DAB kit (Vector Laboratories) according to the manufacturer’s instruction. For CH25H and perilipin staining, 5 μm paraffin-embedded tissue sections were incubated overnight at 4 °C with CH25H (1:50, Santa Cruz Biotechnology) or perilipin (undiluted, OriGene) primary antibody and stained with Tyramide SuperBoost Kit (Thermo Fisher) according to manufacturer’s instruction. Nucleus counterstaining was performed with NucRed 647 (Thermo Fisher).
+ 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!