***** To join or leave the confocal microscopy listserv or to change your email address, go to: https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1 Post images on http://www.imgur.com and include the link in your posting. ***** Hi kurt et al. I agree with these solutions however the problem so far in testing is the fluorescent proteins, I am sure the alexa dyes will be fine... the problem is FP's in a non-aqueous mount. We will work on it and report back. I was hoping that there was a recipe I had missed that the whole world of fish cognoscenti uses, it would appear for the most part that they just breed more fish : - ) Simon C. Watkins Ph.D, FRCPath Distinguished Professor and Vice Chair Cell Biology Professor Immunology Director Center for Biologic Imaging University of Pittsburgh Bsts 225 3550 terrace st Pittsburgh PA 15261 [log in to unmask] Www.cbi.pitt.edu phone:412-352-2277 -----Original Message----- From: Kurt Weiss <[log in to unmask]> Sent: Thursday, April 7, 2022 10:31 AM To: [log in to unmask]; Watkins, Simon C <[log in to unmask]> Cc: Kurt Weiss <[log in to unmask]> Subject: Re: long term mounting of zebra fish embyros Hi Simon, I would echo Philip's suggestion of some form of tissue clearing media, especially an organic solvent such as Methyl Salicylate. Alexa dyes indeed remain stable for a long time in this dehydrated environment. My main concerns would be (1) stability of the coverslip sealant in contact with the organic solvent - though one can't argue with the noted 10-year results so perhaps nail polish is more solvent resistant that I thought. Otherwise, UV cured glue such as Bondic seems quite solvent resistant. And (2) the lenses will not likely be assuming the mounting medium has that high of a refractive index (ca. 1.55) so deep imaging at high NA will likely result in some aberrations, but you should be fine at lower NA/mag suitable for samples of that size. My long winded advice on clearing is here: https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41596-021-00502-8&data=04%7C01%7Csimon.watkins%40PITT.EDU%7Cac6ee68be846445e9da508da18a345d0%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637849386923619990%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=i37mzbbNr%2BWUWBqYrFkYIMyKAabidx9%2Ba9ZDcfeYrj8%3D&reserved=0 Another implementation for long-term mounting of zebrafish was done for a museum install of a light sheet microscope: https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fjournals.plos.org%2Fplosone%2Farticle%3Fid%3D10.1371%2Fjournal.pone.0161402&data=04%7C01%7Csimon.watkins%40PITT.EDU%7Cac6ee68be846445e9da508da18a345d0%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637849386923619990%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=niNWOKK9ty6p%2BPvqj4jUfOKCKBkPJ7YWE%2FcqrNZPSKs%3D&reserved=0 . The main tricks from the methods were nanobody boosters and Vectashield. However, the light sheet specific mounting and 2-month stability (likely due to aqueous environment) may not be in line with what you're looking for. If you go this route I can probably dig up a more detailed protocol. Good luck, Kurt _______________ Kurt Weiss, PhD Biochemistry Optical Core Director UW-Madison Biochemistry Dept. 440 Henry Mall, Rm B1417 Madison, WI 53706 (608) 263-2436 [log in to unmask] https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fboc.wisc.edu%2F&data=04%7C01%7Csimon.watkins%40PITT.EDU%7Cac6ee68be846445e9da508da18a345d0%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C637849386923619990%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=DZ%2FjJbcM1iRBOQ%2FXKHwNhJI3tcHJr7TnoNfZbwpDuv8%3D&reserved=0
