Materials and Reagents

Raw264.7, MCF-7 or Hela cells
DPBS (Life Technologies, Invitrogen^TM, catalog number: 14190-250 )
FBS (Atlanta Biologicals, catalog number: S11110H )
Anti-fade mounting medium: e.g. ProLong Gold Antifade Mountant (Life Technologies, Invitrogen^TM, catalog number: P10144 ) with DAPI (if nuclear staining is needed) or without DAPI
Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
General chemicals (Sigma-Aldrich)
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Tween 20 (Sigma-Aldrich, catalog number: P2287 )
4% paraformaldehyde-freshly-prepared (5 ml) (see Recipes)
Antibodies (see Recipes)

Equipment

Round cover slips (Thermo Fisher Scientific)
24-well plate
Fluorescence microscope

Procedure

Grow cells on round cover slips in 24-well plates to 30-60% confluence. Do not overgrow because it will be difficult to distinguish cell components when stained.
Wash cells with 500 μl/well DPBS twice, then add 250 μl/well 4% paraformaldehyde for 15 min at room temperature (RT).
Remove paraformaldehyde, wash the fixed cells with 500 μl/well PBS + 3% FBS for 3 times.
Permeabilize cells with 250 μl/well DPBS + 0.2% Triton X-100 for 5 min, then wash with 500 μl/well DPBS + 3% FBS for 3 times.
Block with 500 μl/well DPBS + 3% FBS + 0.5% Tween 20 for 1 h at RT.
Remove the blocking buffer, add 250 μl/well primary antibodies and incubate at RT for 1 h.
Remove the antibodies, wash with 500 μl/well DPBS + 3% FBS in 5 min for 3 times.
Add 250 μl/well fluorescent secondary antibodies and incubate at RT for 30 min.
Remove the antibodies, wash with 500 μl/well DPBS + 3% FBS in 5 min for 3 times.
Place a small drop of anti-fade reagent on a glass slide, then get the cover slip from the well and put it face down on the drop, push it tightly and attach to the glass slide.
Leave the slide in the dark for 5 min to let it dry.
The slide now it is ready to observe under a fluorescence microscope.

Recipes

4% paraformaldehyde-freshly-prepared (5 ml)
0.2 g paraformaldehyde powder + 5 ml DPBS + 50 μl 1 N NaOH
Incubate at 65 °C
Vortex several times to dissolved completely
Cool at room temperature then add 4 μl HCl
Mixed completely
Antibodies
Diluted in DPBS + 3% FBS + 0.5 Tween 20
Primary antibodies: 1:100-1:500 (depends on individual antibodies)
Fluorescent secondary antibodies: 1:800

Acknowledgments

This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. This protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA [Chen et al. (unpublished)].

References

Agrawal, S., van Dooren, G. G., Beatty, W. L. and Striepen, B. (2009). Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins. J Biol Chem 284(48): 33683-33691.