Metamorphosis protocol

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L-Thyroxine (T2376) from Sigma- It is on the chemical shelf in Gel room. Small bottle (brown glass- light sensitive- 10 cm size) sitting together with all other small bottles. If you dont find it or trust it then order another one. This one was first opened on 2nd Oct 2012 and I used it last on 06 Nov 2012 with successful results. DMSO (cell culture grade)- I usually take a 10 ml glass vial from cell culture (ask Kerstin or Regina). Use whatever you want to use and freeze the rest in epis at -80. For my experiment, I used frozen -80 DMSO and it worked fine. Prepare a fresh 10 mg/ml stock of Thyroxine in DMSO. I would weigh an empty 2 ml epi and then I would just add a bit (a couple of mg powder thyroxine) in it and weigh it again. Open Thyroxine bottle under the HOOD and take a bit with help of spatula and put it in the epi. Close the epi under the hood and then take it out and weigh it. Add DMSO according to the weight to make the 10 mg/ml stock under the hood. Once diluted, you can work on the bench. Dilute this (10 mg/ml) stock to 1 microgram/microliter (1 μg/μl) with DMSO. I would take 100 μl of the stock (10 mg/ml) and dilute into 900 μl of DMSO to obtain 1 μg/μlworking solution. Inject (1 μg /ul- working solution) in upper thoracic cavity (lung region) under the skin. Inject 1.5 - 2.0 microliter (μl) per gram body weight of the animal using a gas tight hamilton syringe (Hamilton Cat # 1702) with a 30 G 1/2 inch 0.3 x 13 mm (BD) disposable needle. I did not see a difference in success rate of metamorphoses when I injected 1.5 or 2.0 μl per gram of animal weight. BUT keep it 1.5 μl per gram to be on the safe side and not get lethality if injecting precious animals. The hamilton syringe (25 μl volume) is in my drawer (labeled for thyroxine only). It’s the same make and type that we use for tamoxifen injections. SO Check the label on the syringe and on the box that it says for “Thyroxine only”. Inject WITHOUT fast green. I had 80 % success (conversion) when I injected without Fast green. Don’t waste your time injecting with Fast Green. I did that already many a times. After injection, keep the animals in hydration chamber for 30-45 minutes and then return them to fresh water. I would not change water of the animals for a couple of days unless it needs to be changed. Use a separate net for changing water and keep the boxes and net separate that comes in contact with these animals. Check the animals atleast twice a day to see if they are doing OK. It can change from one day to the next and one can find surprise dead animals with blisters on their abdomen from one day to the other. In my last (most successful experiment), I injected 6 animals in total. All six animals survived the injection.

The animals ranged in size from 10 to 10.5 cm and weighed between 10.88 g to 12.49 g. 3 were injected with 1.5 μl per gram and 3 were injected with 2.0 μl per gram body weight of the 1 ug/μl working solution. 2 animals from each group metamorphosed and 1 animal from each group did not metamorphose or even showed signs of reduced gills etc. You will observe reduction in gills as first sign after 7-10 days. As soon as gills start to regress, keep an eye on them and once gills are getting very small, lower the water level to a point that only the animal is dipped in it completely but has a chance to stick its head out and breath if it wants to. Once gills are completely gone. Keep the level of water as stated above (just enough that animal is completely covered in it). Put an inclined rock (not smooth- as you can see what I have put in the previous ones) and tilt the box to have a rocky beach for the animal to climb up on. Here is an example of two different successful injections: 1.5 μl per gram body weight weight of animal: 11.97 grams Injection volume: 17.9 μl 2.0 μl per gram body weight weight of animal: 11.88 grams Injection volume: 23.76 μl Once the process of gills reduction starts, it goes very fast and the eyes pop out and dorsal fin also regresses. They might not eat much while metamorphosing but keep on feeding them everyday. Once they metamorphose completely, they will start eating and digesting food. They will shed their skin quite often and need to be cleaned more than their unmetamorphosed counter parts.