Special Requirements for Different Cell Types
Cell Types used for Confocal Microscopy in this facility:
- 293T (Human Kidney cell line)
- U373 (Human brain astrocytoma cell line)
- A172 (Brain glial cell line)
- HuVec (Human umbilical vein endothelial cell)
- Trypanosoma brucei
- Bacillus subtillus
Fixation protocol for 293T cells (tried and tested)
Fix in ethanol (95% v/v) at 4oC for 1 hour (minimum). Then re-hydrate in PBS.
If you are using GFP do not fix in ethanol, fix with paraformaldehyde
Fixation protocol for HuVec
Fix in ice-cold methanol (100%) at -20oC for 15 minutes.
Fixation protocol for Trypanosoma Brucei
Fix with paraformaldehyde (3% w/v, final) for 10 minutes at 0oC.
- If you seed your cells for 72 hours rather than 48 hours the cells attach better to the coverslip and they look better morphologically.
- If you are using PI for the nuclear stain, add it as the last incubation in the blocking buffer at a concentration of 2 mg/ml and incubate for 2 minutes.
- If you are using dapi/Hoechst for the nuclear stain, add it to the mounting media.
- Poly- L -lysine is not required for HuVec to adhere to the coverslips. These cells adhere very well to plain chamber slides.