Use high quality nanopure water to make all of the solution and use the new petri dishes. Gently shaking the whole incubations is recommended.
- You can initially will gel stained with Coomassie blue and then destained with 10% acetic acid for storage. (Keep the wet gel by wrapping parafilm around the plate.)
- When you are ready to make your silver stain, fix blemishes by 50% methanol and 0.038% formaldehyde (formalin concentrated should be 38%). Please note formaldehyde and methanol are both toxic and should be used in a fume hood.
- After at least 1 hour (or overnight), aspiration of fixing solution, and wash 3-4 times with water more than 30 minutes.
- Meanwhile, prepare the stain. Make 5.25 ml of 0.36% sodium hydroxide. Add 350 ul of concentrated ammonium hydroxide (added in a fume hood such as ammonium hydroxide is volatile). Then, while stirring, add 200 mg / ml solution of silver nitrate before it turns completely yellow / brown. (It should take approximately 300 ul of silver nitrate.) If the solution turns yellow / brown, you have to start again. Make up to 25 ml.
- Change the water stains, and stain for 15 minutes.
- Wash for 5 minutes with water.
- Develop with the developer containing 125 ml of water, 0.625 ml of 10 mg / ml of citric acid, and 63 ul of 38% formaldehyde. It should take less than 10 minutes.
- Stop the reaction with several washes of 45% methanol, 10% acetic acid. Take pictures or dried gel. Try to keep the plate in the dark if you do not see or take a picture directly.