Animals

Biological Electron Microscopy Sample Preparation Protocol for Rabbit Corneal Tissue

1. Animal Tissue Sample Collection:
New Zealand rabbits were euthanized, and corneal tissues from different groups were collected. Sampling was performed within 1-3 minutes post-euthanasia. Corneal tissue samples were trimmed to approximately 2mm × 2mm in size and made as thin as possible. If immediate trimming was not feasible, samples were first placed in electron microscopy fixative for approximately half an hour to allow hardening before trimming and subsequent post-fixation. (Failure to adhere to this time frame may result in incomplete fixation, rendering subsequent experiments impossible. This step is critical.)
During collection, care was taken to avoid mechanical damage such as forceps compression, and a sharp blade was used to prevent crushing the tissue.
Immediately after dissection, corneal tissues were immersed in electron microscopy fixative and fixed at room temperature in the dark for 2 hours. They were then transferred to 4°C for storage (ensure samples do not freeze). Samples can be stored under these conditions for up to approximately one month. 

2. Double Fixation Procedure:

• Primary Fixation: Tissue pieces were immersed in 2.5% - 3% glutaraldehyde fixative (prepared commonly in 0.1M phosphate or sodium cacodylate buffer) and fixed at 4°C for 2 hours to overnight.
• Rinsing: Samples were thoroughly rinsed 3-4 times with the same buffer used for fixation, for 15-20 minutes each, to completely remove glutaraldehyde.
• Post-fixation: Tissues were fixed in 1% osmium tetroxide (OsO₄) solution at 4°C or room temperature for 1-2 hours. Osmium tetroxide fixes lipids and enhances the contrast of membrane structures.

3. Dehydration and Infiltration:

• Dehydration was performed using a graded series of ethanol or acetone (e.g., 30%, 50%, 70%, 80%, 90%, 100%), with each step lasting 15-30 minutes.
• Following dehydration, propylene oxide was used as a transition solvent. Tissues were then infiltrated with mixtures of propylene oxide and epoxy resin (e.g., Epon 812) in varying ratios, culminating in pure resin infiltration.

4. Embedding, Sectioning, and Staining:

• Tissue pieces were placed in embedding molds and polymerized in a 60°C oven for 24-48 hours to form hard resin blocks.
• Ultrathin sections of 50-70nm were cut using an ultramicrotome and collected on copper grids.
• Double Electron Staining: Grids were stained first with uranyl acetate, followed by lead citrate, to enhance the contrast of cellular structures.