A carefully pinned STNS
After each dissection was completed the tissue was carefully pined as flat as possible into the fix dishes, covered with saline, and labeled in both in the master book and on the dish. All of the tissue samples collected during that day would then be fixed overnight. The fix solution consisted of 4% Paraformaldehyde, The entirety of the Paraformaldehyde ampoules’ (10ml, pre-diluted) was mixed with 30ml of PBST to total 40 ml of liquid. Using gloves, the saline was poured out of each of the fixing dishes and was replaced with the 4%PARA solution. The tissues would sit in the fix solution overnight. After the fixation process each tissue was rinsed 5 times every half hour using PBST. One half hour after completing the rinses, each tissue was assigned one of three test antibodies: a-CLDH (calcitonin like diuretic hormone), a-melatonin receptor, a-melatonin. For each of these antibodies a solution was made and placed into the Eppendorf tubes.
The primary antibodies were comprised of 10% Normal Donkey Serum (NDS), phosphate buffer solution with tricane-x (PBST), and antibody. The melatonin antibody was made with a ratio of 1:500, the melatonin receptor antibody was made with a ratio of 1:300, and the calcitonin-like anti-diuretic hormone (CLDH) was made at a ratio of 1:1000. These ratios represent the amount of given antibody to the total volume.
The solution for each antibody was then placed carefully into Eppendorf tubes, using extreme care to not cross contaminate. For eyestalks the tubes were filled halfway, while the other tissues (P.O and S.T.N.S) were filled all the way. Each tissue was next carefully removed from the fix dishes using microscopes to properly remove the pins and placed into the properly labeled Eppendorf tubes. The tissue then sat in the primary antibody for 3 days. Next a second rinse was conduced rinsing the tissue 5 times in PBST every half hour. One half hour after the last rinse the tissues were moved into the same secondary antibody a- donkey anti rabbit solution:
The secondary antibody was made in a similar fashion to the primary antibodies. We used a 1:300 ratio of antibody to total volume, again with 10% comprised of NDS, antibody, and the remaining volume of PBST. As mentioned, regardless of the primary antibody used, all tissues received the same secondary antibody.
The secondary antibody was extremely sensitive to light, and both the Eppendorf tubes and the solutions need to be covered with tinfoil at all times possible. The tissues would sit in the secondary antibody overnight. In the morning a final rinse was conducted, 5 rinses, every 30 minutes in PBS and one hour after the final rinse the tissue was removed from the Eppendorf tubes and mounted.
Slides were prepared using 2-3 drops of Vectashield with or without DAPI. Tissue samples were added and covered with a clear glass cover slip sealed with clear nail polish. They were labeled according to their tissue type, species, and primary and secondary antibodies.
Fluorescence and brightfield microscopy
After mounting, we waited an appropriate amount of time for the slides to dry. All slides were initially looked at under a Zeiss Axiovert 200 fluorescence microscope. We viewed a total of 59 stained slides under the fluorescent microscope and chose selected slides to be imaged in more detail using an Olympus Fluoview 1000 confocal microscope.