I want to collect leukocytes from fish skin, can anybody recommends me a protocol, I know that using a dispase could be helpful but I need more details. Thanks :)
Hi,
This would be theoretically a bit tricky. Most unstimulated immune cells should be circulating in the blood stream or residing quietly in client tissues (e.g. liver) in a "stand-by" mode, rather than being poised to "act" in the skin.
However, induction of a physical wound will send signals to distant leukocytes, such that they migrate to the wound site and therefore become concentrated there.
An unwanted effect or consequence of this is that the recruited immune cells would then likely get activated and start to generate and release immune effectors, altering their own gene expression profiles.
See this Nature paper a few years back on wounding model in zebrafish. Make a tail in cut will help you recruit leukocytes to a site. H2O2 was said to be a principal recruitment signal.
http://www.nature.com/nature/journal/v459/n7249/abs/nature08119.html
Likewise, certain microbial infection in theory should increase the immune cell density in skin through recruitment.
Nai-Kei wong, thanks for your prompt reply, You are right with the migration of leukocytes to a wounded area to do a function there and how it would be changed a little bit than the quiescent state, but let me tell you the whole story, we immunized our fish with I.p injection vaccine to quantify the mucosal antibody, fortunately we found an increase in specific mucosal Ab against our vaccine Ag, now we want to find a relationship between the secreted mucosal Ab and the ASC in dermal leukocytes, some protocols (one of them in nature) mentioned the use of dispase to isolate the skin leukocytes, but as you know usually you need some details and trials and so I am asking, If somebody has an experienced hand with this.
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