I am doing a project on trying to stain and image the neuromuscular junctions of drosophila leg muscles in great detail (with the possibility of imaging the boutons as well). So I have got two questions pertaining it:
1. I am going to fix the leg tissues in PFA but I have got contradicting protocols whereby some instruct to fix overnight at 4 degrees celsius while some simply instruct to fix at room temperature for 20-23 mins. Does this really make a difference or would doing the latter be sufficient? Also, I would like to know if fixing at a low temperature overnight might create any negative consequences on the results.
2. We have tried imaging the NMJs of adult drosophila legs before, whereby on anti-HRP was used as the only primary antibody. Several papers that I have read through have also mentioned anti-Dlg and anti-Brp as good candidate antibodies to visualize the boutons with greater precision. However, at the moment, we do not have anti-Dlg and anti-Brp so I am thinking of trying out another antibody to stain. Would anti-vGlut be a good choice of primary antibody to visualize the boutons?
Thank you in advance!
1) As proteins and other groups react with formalin and that formalin takes many hours up to days to make all crosslinks, macro,molecules are prone to conformational changes.
2) Autolytic processes by intracellular and extracellular lysosmal enzymes occur hardly at 4 oC. At 20 oC they have a lot of residual activity. They will also fixed by the formalin and their active centres will be blocked.
Both items are important to preserve the conformation of e.g. antigens and remain nucleic acid targets accesible for e.g in situ hybridisation (using correct pretreatment).
Take in mind that formalin fixation takes more than a week at rT to be completed. So, when you use 4% buffered neutral formalin at 4 oC your tissue still rather underfixed but stable enough in tissue processing. It remains accesible for all types of post-embedding analyses,
Elizabeth Van Pelt-verkuil Alright, thank you very much! I unfortunately do not have much time to do a fixation over a week, I will do it at room temperature for about 25 minutes as suggested by a protocol that I am looking at.
No; thats NOT what I recommend;
I recommend 24 hrs at 4 oC. NOT fix for a week. I only told that to illustrate that after a day, tissue will be underfixed. I.e. Not all possible crosslinks have been formd between recative groups in the tissue and fomalin
Elizabeth Van Pelt-verkuil Oh I see! I am sorry I misunderstood that.. But thank you anyways!
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