NB: All slide washes are carried out in Coplin jars, while permeabilisation, blocking and antibody incubations are carried out ‘in-well’ on the slide (in the dark under cover to minimise evaporation and 2° antibody fading)
Protocol 1: ‘Drying & PFA fixation’
This is a particularly useful approach, if you’re planning to analyse a large number of cell lines or several time points, as multiwell slides can be used without risk of interwell exchange, and dried cells are stable for several days in the hood.
- Add 0.5 ml ice cold 4% (v/v) paraformaldehyde in PBS to 0.5 ml bloodstream form T. brucei culture (~5×105 cells), invert gently several times and place on ice for 15 minutes
- Wash: 1 minute at ~4,500 g, remove 900 μl supernatant and gently resuspend the cells in the remaining supernatant, add 900 μl cold PBS; repeat PBS wash and resuspend in cold 1% (w/v) BSA in H2O, repeat centrifugation and resuspend in residual ~20 μl supernatant
- Spread 2 μl cell suspension onto a well of a multiwell slide, check the density using an inverted microscope (add more cell suspension, if necessary), and air dry in the hood for ~30 minutes (slides can be left for several days prior to continuing the procedure, if required)
- If localising intracellular antigens, permeabilise the fixed cells in 0.5% (v/v) Triton X-100 in PBS for 20 minutes, otherwise go straight to step 6
- Wash three times five minutes in PBS
- Block with 50% (v/v) foetal bovine serum in PBS for 10 minutes, remove and replace with the 1° antibody in 3% (v/v) FBS (in PBS) for 45 minutes
- Wash three times five minutes in PBS
- Incubate with 2° antibody in 3% (v/v) FBS (in PBS) for 45 minutes
- Repeat the washes in (7), counter-stain with DAPI in Vectashield (Vector Labs) or any suitable anti-fade mountant, and seal the cover slip/slide edges with nail polish.
NB: In the case of procyclic forms, permeabilise in 0.1% Triton X-100 for 10 minutes; alternatively, incubate in ice cold methanol for 10 minutes (instead of or in addition to PFA; this works for bloodstream and procyclic form T. brucei – see Protocol 2).
Protocol 2: ‘Drying & Methanol fixation’
Not all epitopes are amenable to paraformaldehyde fixation; a good alternative is treatment with methanol
- Pellet ~106 cells at 4,500 g and resuspend in 1 ml PBS; centrifuge again and resuspend the cells in ~20 µl residual PBS
- Spread 2 μl cell suspension onto a well of a multiwell slide, check density using an inverted microscope (add more cell suspension, if necessary), and air dry in the hood for ~30 minutes
- Place the slide in a methanol, pre-chilled to -20°C, for 10 minutes
- Transfer to PBS and continue from step (6) in Protocol 1.
NB: There is no need for a separate permeabilisation step in this protocol, as methanol treatment delipidates the cells, effectively permeabilising the cell membranes.
Protocol 3: ‘Settling & PFA fixation’
Because cell-slide attachment relies on pre-treating the slides with poly L-lysine rather than drying, the cells retain better morphology, making this is a good approach if cell shape is critical, for instance to enable 3D localisation of intracellular antigens within the parasite by confocal microscopy
Slide preparation: poly L-lysine coating
- Clean the slides by soaking in 0.25 M HCl for 5 minutes
- Rinse thoroughly in distilled H2O – three times
- Place in a 10:1 dilution of poly L-lysine (Sigma) for 5 minutes (the solution can be stored at 4°C and used for up to 3 months)
- Dry overnight on the bench at room temperature (or for ~1 hour at 56°C).
- Slides can be stored for a year on the bench.
- Fix cells as in Protocol 1
- Wash: 1 minute at ~4,500 g, remove as much supernatant as possible, add 1 ml PBS, repeat centrifugation and resuspend in up to 100 μl supernatant
- Transfer 30 μl to one well of a pre-treated multi-well slide
- Allow the cells to settle for 15 minutes, draw the remaining liquid off the slide from the edge of the well with a paper towel, and place the slide gently in PBS for 5 minutes; continue from step (4) in Protocol 1.
NB: It is important to remove as much formaldehyde as possible from the cell suspension prior to placing on the slide, otherwise cell adhesion to the treated slides may be inhibited.