Fixation for Cryosectioning Protocol
9 January, 2023
The aim of this protocol is to provide instructions for fixation for cell-laden 3D bioprinted constructs for cryo-sectioning.
- Cell laden 3D bioprinted constructs
- Formaldehyde solution (PFA) 36.5–38% from Merck, SKU: F8775-25ML
- Crosslinking Agent (for alginate containing bioinks)
- 30% Sucrose in PBS
- Hank’s Balanced Salt Solution (HBSS+/+)
- Phosphate-Buffered Saline (PBS)
- Embedding cassettes
1. Preparation of 4% PFA
For alginate containing bioinks, the 36.5-38% PFA can be diluted in Crosslinking Agent with 50 mM CaCl2, to minimize the risk of the construct dissolving in the PFA. If not using alginate containing bioinks, dilute the PFA with PBS.
For suggested PFA, which is 36.5-38%, mix 1.1 mL of PFA with 8.9 mL PBS or 50 mM Crosslinking Agent to receive 10 mL 4% final PFA concentration.
Submerge the 3D bioprinted samples in the 4% PFA and fix the constructs for 2-24 h at room temperature. Alternatively fix the samples 1-2 hr in RT, transfer samples to 4°C and continue the fixation for 24-48 hr.
Note: Adjust the time according to experimental needs.
Wash the constructs 2 x 10 min with HBSS+/+ at room temperature.
Add enough HBSS+/+ to completely cover the constructs. Seal the vessel with parafilm and incubate at 4°C for 45 min.