Lately I have been trying to perform immunofluorescence staining for connexins in primary rat cardiac cells but I donot visualize connexin gap junctions at all in my samples. I compared it with images illustrated in research papers and I am wondering what is going wrong. Can someone help me out with this.
Thanks in advance
Hi Krishna
First of all, make sure you have the right antibodies (right species and right isotypes). Second, use a positive control. I think brain tisue is a good positive control for connexin43. If you couldn't get any results in other tissues, then check your antibody dilution. IHC protocols can be tricky sometimes as they may strongly vary lab to lab. If you are using polyclonal antibody, use 1/100 to 1/1000 and for monoclonal use 1/1000 and above.
Finally, check your microscope with control fluorescent dyes to see if it actually works! :)
Wish you good luck, and please update me with your results.
Thanks Daniel and Dakshesh for your insights. I will update once the results are there.
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