Summary
This protocol covers OCT embedding, cryosectioning, staining and imaging of fixed LunaX hydrogel constructs. It supports histology, histochemistry, immunohistochemistry and immunofluorescence for high-resolution analysis of cellular structures, proteins and extracellular matrix components.
What you’ll do
- Fix LunaX hydrogel constructs and embed them centrally in OCT compound.
- Prepare approximately 10 µm cryosections on positively charged slides.
- Fix sections with ice-cold acetone and condition them in BaCl₂/Tris-HCl buffer.
- Apply histological, histochemical, immunohistochemical or immunofluorescence staining.
- Image sections and quantify marker intensity, localisation or stain-positive area.
Protocol at a glance
- Fixation: 4% PFA for 2 hours at room temperature.
- Embedding: OCT compound, frozen on dry ice or in liquid nitrogen.
- Sectioning: Approximately 10 µm using a cryostat.
- Section conditioning: Ice-cold acetone for 10 minutes, followed by 50 mM BaCl₂ in 100 mM Tris-HCl for 1 hour.
- Readout: Brightfield histology, chromogenic IHC or fluorescence microscopy.
Expected outcome
Processed cryosections should retain construct morphology and accept the selected histological or antibody-based stain. Target-specific signal and counterstains should remain clearly resolved, while isotype, no-primary and secondary-only controls show minimal non-specific labelling.
Critical parameters
- Position the construct centrally in OCT and use positively charged slides to support section retention.
- Air-dry sections before and after ice-cold acetone fixation.
- Retain the BaCl₂/Tris-HCl conditioning step, which supports section integrity and antigen accessibility.
- Optimise antigen retrieval, blocking and antibody conditions for each target.
- Include appropriate positive, isotype, no-primary and secondary-only controls.
