Special Requirements for Different Cell Types
Cell Types used for Confocal Microscopy in this facility:
1. 293T (Human Kidney cell line)
2. U373 (Human brain astrocytoma cell line)
3. A172 (Brain glial cell line)
4. HuVec (Human umbilical vein endothelial cell)
5. Trypanosoma brucei
6. 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.
Notes:
- 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.
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