Cytometry Lab

Sample Preparation

This is a general method for immunofluorescence of cells fixed on coverslips, and can be modified according to your experiments.

 

Solutions used:

PBS	Phosphate buffered saline
PBST	Phosphate buffered saline containing 0.1% Tween-20
3.2% PFA	200 mls PBS; 6.4g PFA (paraformaldehyde, Sigma)
150 mM Gycine/PBS	200mls PBS; 2.25 g glycine

 

Growing cells on coverslips

(sterile conditions apply)

• If possible, use no. 1.5 coverslips as they provide the best optical performance when used with the lenses on our microscope

• To prepare coverslips, soak in 70% EtOH, place into 10 cm petri dishes or multiwell plates and allow for EtOH to evaporate for ~30 min

• Cells can be added to these coverslips and grown to the desired confluency. Transfections can be performed on these cells if required

 

Growing cells on sterilized coverslips

 

Fixing cells

• Wash cells 2X in PBS

• Fix with 3.2% PFA, room temperature (RT) for 15 min.

• Wash 2X in PBS

• Cells can be kept in PBS at 4°C for up to 2 months

 

Processing

• Cover cells with 150mM glycine in PBS, RT 15 min (to block amine groups, reduce autofluorescence)

• Wash 2X in PBS

• Incubate cells in PBST for 15 min, RT (to permaeabilize cells)

• Block cells in PBS 10% non-immune serum for 30 mins, RT (use non-immune serum that matches the animal that your secondary antibody was generated in, if possible)

• Wash 3X in PBST

• Incubate with 1° Antibody 1-2 h, RT (Ab made up in PBST). Approx volumes: 50 ml/13 mm coverslip 75-100 ml/18 mm coverslip

• Wash 3X in PBST

• Incubate cells with 2° Antibody, 30m, RT (Ab made up in PBST)

• Wash 5X in PBST

• Optional step: Counterstain with DAPI, 15 min, RT (1/20,000 dilution of a 10 mg/ml stock)

• Mount cells with anti-fade mounting media and leave overnight before CLSM

 

Mounting cells on slide