Summary
This protocol adapts standard tissue histology methods for paraffin embedding and sectioning of LunaX hydrogel constructs. It covers fixation, graded dehydration, clearing, paraffin infiltration, embedding and microtome sectioning for downstream histology, special stains and immunohistochemistry.
What you’ll do
- Fix LunaX constructs in 4% paraformaldehyde.
- Dehydrate samples through a graded ethanol series.
- Clear constructs in xylene and infiltrate them with molten paraffin.
- Orient and embed constructs for the intended sectioning plane.
- Prepare 5–10 µm sections on positively charged slides for downstream staining.
Protocol at a glance
- Fixation: 4% PFA for 2 hours at room temperature.
- Dehydration: 30%, 50%, 70%, 90% and two changes of 100% ethanol.
- Clearing and infiltration: Sequential 1-hour xylene and paraffin stages, with paraffin held at 60 °C.
- Sectioning: 5–10 µm using a rotary microtome.
- Slide preparation: Float at 40–45 °C, then dry at 60 °C for 30–60 minutes.
Expected outcome
Properly processed constructs should yield intact paraffin sections with preserved hydrogel architecture, minimal tearing or wrinkling and reliable adherence to positively charged slides. Sections should be suitable for H&E, special stains and immunohistochemistry.
Critical parameters
- Complete each dehydration and clearing stage; incomplete reagent exchange causes tearing and poor morphology.
- Handle constructs gently and orient them for the intended sectioning plane.
- Apply vacuum during fixation or paraffin infiltration when improved penetration is needed.
- Extend processing times for larger constructs.
- Confirm xylene-substitute compatibility with LunaX ECM and planned downstream stains.
