Summary
This protocol describes whole-mount immunofluorescence staining of intact 3D cultures encapsulated in LunaX hydrogels. It supports visualisation of nuclear, cytoskeletal, cell-surface and intracellular targets without sectioning, using widefield or confocal fluorescence microscopy.
What you’ll do
- Fix intact LunaX constructs with 4% paraformaldehyde.
- Permeabilise and block samples to reduce non-specific antibody binding.
- Incubate constructs sequentially with primary and secondary antibodies.
- Add optional phalloidin and DAPI counterstains.
- Acquire widefield images or confocal z-stacks through the construct depth.
Protocol at a glance
- Sample: Intact LunaX ECM-encapsulated 3D cultures.
- Fixation: 4% PFA for 45 minutes at 4 °C.
- Primary antibody: Overnight at 4 °C, followed by at least 8 hours of washing.
- Secondary and DAPI stages: 2 hours each at 4 °C with extended washes.
- Readout: Widefield fluorescence or confocal z-stack imaging.
Expected outcome
Successfully processed constructs should show distinct target-specific fluorescence with low background. Nuclear and optional cytoskeletal counterstains should remain clearly resolved through the construct, enabling confocal z-stacks and maximum-intensity projections across a defined imaging depth.
Critical parameters
- Optimise antibody dilution, fixation and permeabilisation for each antigen and cell model.
- Incubate primary antibody overnight and use long washes with at least three buffer changes.
- Keep antibody and DAPI stages sequential to minimise background signal.
- Protect samples from light after fluorophores are introduced.
- Reduce or omit Triton X-100 for cell-surface or permeabilisation-sensitive epitopes.
