To begin staining the fixed retinal organoids, or ROs, for transmission electron microscopy, use a pipette to remove the osmium acid previously added to the organoids in the microcentrifuge tube during post-fixation. Then, wash the ROs with 0.01 molar PBS at pH 7.4 three times for 10 minutes each, followed by washing with deionized water three times for 10 minutes each. Remove the last deionized water wash, replace it with about 150 microliters of uranium acetate and stain for one to two hours at room temperature.
Next, in a fume hood, remove the uranium acetate and replace it with one milliliter of 50%acetone to dehydrate the samples for 10 minutes. Then, perform gradient dehydration using 70%80%90%and two times 100%acetone successively, each for 10 minutes. After the gradient dehydration, discard the residual acetone and add approximately 150 microliters of a mixture of acetone and Epon-812 resin.
Place the tube in a 37-degree Celsius oven for one hour. After removing the previous acetone-resin mixture, replace it with a different composition of acetone and Epon-812 resin mixture. This time, incubate the tube at 37 degrees Celsius overnight.
Finally, transfer the retinal organoids into a new tube containing approximately 500 microliters of pure epoxy resin, carefully. And incubate at 45 degrees Celsius for one hour before proceeding with organoid embedding.
