I have no background on neuroscience but I need to evaluate the neuropathy side effect of a treatment in my project. I did toluidine blue staining for the sciatic ganglions. Based on literature, I got the point that degranulation of mast cells and dissolution of nissl bodies can be as markers for neuropathy. But it's still hard for me when it comes to the real data. I appreciate it if you can do me a favor.
Pictures are attached. Group 1 and 3 are blank (saline) control and a known neurotoxic treatment control respectively.
Firstly it seems that your staining is marking a few other structures, it also seems like your fixation has caused a few artifacts which has destroyed the structure of your cells. What protocol did you happen to use for this staining? From what I see, you should have a dark blue-purple colour. Are you sure about the pH perhaps?
Secondly, if you'd like to detect devgranulation of mast cells, you will probably need to work at a higher magnification, though it might be OK
http://www.ijdr.in/article.asp?issn=0970-9290;year=2012;volume=23;issue=5;spage=695;epage=696;aulast=Reddy
Your staining should look more like the attached URL. That is, it should be clear and consistent throughout the sample. It doesn't look like you are far off though. Unfortunately, molecular biology can be more of an art than a science at times. If you are doing the staining yourself I would reach out to someone in a biochemistry or molecular biology department near you to see if they can help you improve your results.
http://www.bing.com/images/search?q=blue+staining+of+the+sciatic+ganglions&view=detailv2&&id=1ADA20A0BFF605357F2158C9A6CF4878D52BBDC4&selectedIndex=16&ccid=ZrC4vdeV&simid=608037382584012194&thid=JN.duyKekhxaAAmZqNq1ke7Ig&ajaxhist=0
Thank you Olivier and Jeffrey. Thank you for sharing the reference and pics. As you said, the staining seems didn't work well. Now I'm gonna send samples to a commercial lab and let them do the staining. What a histochemistry layman I am :(
Don't feel bad. Like I said, it looks like you are pretty close. It is just that for a lot of these biochem/molecular bio processes the difference between a good outcome and a bad outcome can be the difference between o.oo1% chemical x, or 1 second more/less in reagent y, or etc. The process is always the same, but the difference between a good result vs a bad result is almost always either experience or luck. That's one of the reasons I don't do bio anymore. I do miss is though. =o)
..but yeah, if you're not trying to do toluidine blue staining as part of your living then I would let someone else do it.
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