Which method was used to validate Beclin-1 expression in canine tissues?

• A. Immunohistochemistry on serial sections
• B. ELISA
• C. qPCR
• D. Western blot

Answer: (D)

Beclin-1 expression in canine tissues is best validated at the protein level with Western blot. This method directly shows whether the protein is present and confirms its identity by detecting a band at the expected molecular weight after separation by size. By running tissue extracts, transferring to a membrane, and probing with a Beclin-1–specific antibody, you obtain a clear signal only if the protein exists in the sample. The band intensity also lets you compare relative expression across samples, and including a loading control ensures that differences aren’t due to unequal protein amounts.

Other approaches don’t provide the same combination of identification and size confirmation. qPCR measures mRNA levels, which don’t always translate to protein abundance due to post-transcriptional regulation. Immunohistochemistry shows where Beclin-1 is localized within tissue sections but is less reliable for precise quantification and does not confirm the protein’s size. ELISA can quantify protein amounts but typically doesn’t reveal the protein’s molecular weight, and relies on antibody specificity in a potentially more complex background.

So, detecting a Beclin-1 band at the expected size by Western blot gives the most specific and informative validation of its expression in canine tissues.