Classic immunofluorescence protocol
Classic immunofluorescence protocol
Written by Guillaume Jacquemet
Prepare fixation solution:
– PFA 4%
PFA based fixation is a good method to preserve actin stress fibers
- Weight 40g of PFA for 1 L of MilliQ water
- Dissolve the PFA at 37°C in a waterbath (overnight)
– Methanol
Methanol based fixation is a good method to preserve microtubules cytoskeleton.
Use methanol previously cooled at -20°C (store methanol in -20°C until use).
– PFA 4% plus 0.05% gluteraldehyde
PFA supplemented with 0.05% gluteraldehyde is a good method to preserve both actin and microtubules cytoskeleton.
Prepare coverslips:
(If you are using MATTEK glass bottomed dishes you do not need to do this.)
- Boil coverslips 3 times in MilliQ water to clean them
- Put coverslips into a 24 well plate
- Coat your coverslips with FN overnight (or other ligand)
- Block your coverslips with a solution of 10 mg.ml of denaturated BSA (dissolved in PBS)
Prepare cells:
- Trypsin and plate cells
- Fixe cells using the right fixation solution for 20 min (or overnight at 4C)
- Wash 3 times with PBS
At that stage, fixed cells can be stored in PBS at 4°C until needed.
Stain cells
- 3-4 min 0.5% triton (only if intracellular stainning)
- Quenching with 0.1M filtrated glycin (at least 20 min)
- Blocking step with 3% non denaturated BSA (optionnal, trying without can improve stainning)
- Primary antibody diluted in PBS or 3% BSA (10ug.ml is suitable for most antibodies), incubation for 45 min at RT
- -Drop technique: 35-50 uL per coverslide
- -24 well plate: 200 uL
- 3 washes with PBS (or with PBS + 1% tween)
- Secondary antibody diluted in PBS or 3% BSA (1/200), incubation for 45 min at RT; protected from light
- 3 washes with PBS, plus one wash with MilliQ water
Mount coverslips on slides
- Clean glass slides with 70% ethanol and let them dry
- Put a drop of monting medium on the glass slide (about 20 uL)
- Mount the coverslides, cells facing the glass slide, into the monting medium
- Let dry overnight in the dark
- Store in the dark at 4°C or at -20°C (long term storage)