title graphic AIF graphic

star.GIF (4493 bytes)Immunofluoresence

This protocol is used by Dr. Maryse Bailly for studying metastatic cancer cells in culture such as MTLn3 and MTC cells and could be modified for other mammalian cell culture systems.

You need to have a sample of the same cells treated with the same protocol at the same time but with one deletion:  no primary antibody.  You must have this no primary antibody control to ascertain whether positive fluorescence is specific binding or nonspecific background.  For double labeling, controls should be included to make sure there is no fluorescent spillover from one channel to another and controls for double immunolabeling should check for crossreactivity of antibodies.

Example protocol for immunofluoresence of dynamic mammalian cell culture

Immunofluorescence / F-actin staining on coverslips or Mattek dishes (MTLn3 cells)
Dr. Maryse Bailly   16 May 2000

Day 1:

  1. Wash coverslips or Mattek dishes with 1 M HCl for 3-5 min. Wash with sterile DPBS (2ml), followed by 95% ethanol (1-2 min), and a final wash in sterile DPBS (keep them in DPBS until you plate the cells).
  2. Plate 200,000 cells per 35 mm dish (usually 2 ml of a 100,000 cells/ml suspension) in complete medium.
  3. Make first part of mounting medium (N-Propyl gallate and glycerol).

 Day 2:

  1. If cell stimulation is needed, remove whole medium and replace with 2 ml MEMH-BSA for 3 h (serum starvation); then stimulate the cells by adding 2 ml of a 10 nM solution of EGF (2x concentrated) in MEMH-BSA at 37oC for desired time.
  2. Remove medium and add 2 ml 37oC 3.7 % formaldehyde in 1 X Fix buffer for 5 min. (Do not put dishes with formaldehyde back in incubator or in the hood but place all plates on styrofoam block to maintain temp). After fixation all the rest can be done at room temp.
  3. Remove fix and add 2 ml of 0.5 % Triton-X100 in 1 X Fix buffer for 20 min.
  4. Remove Triton and rinse 1 time with 0.1 M Glycine in 1 X Fix buffer and then wash in 0.1 M Glycine for 10 min.
  5. Do 5 quick washes with 1 X TBS, pH 8.0; then block/stain with 0.5 uM Rhodamine-phalloidin in 1 % BSA, 1 % FCS in 1 X TBS, pH 8.0. (50-100 ul volume is enough for a 10 mm-well Mattek) in humidified chamber for 20 min. If you do not wish to stain the F-actin, use 5 uM of unlabeled phalloidin instead of the labeled phalloidin (this will stabilize the cytoskeleton).
  6. Aspirate the Rh-phalloidin/TBS and replace by the primary antibody diluted in 1 % BSA in 1 X TBS (50 ul volume is enough). Incubate 1 hour at RT in humidified chamber.
  7. Wash 5 X 5 min with 1 % BSA in 1 X TBS (last wash can be done in TBS alone to get rid of the BSA).
  8. Incubate for 1 h in secondary antibody in 1 % BSA in 1 X TBS (50 ul volume is enough) in humidified chamber.
  9. Wash 6 X 5 min with 1 % BSA in 1 X TBS.
  10. Mount by adding 100 ul mounting medium to well and covering with coverslip. Seal coverslip with nail polish (essential to prevent movement with oil immersion microscope objectives), wrap in foil, and store at 4o c for viewing. If bubbles form in well, seal bottom coverslip with nail polish.

ACTIN_VOXELVIEW.GIF (13549 bytes)Solutions

MEMH-BSA

47.4 ml alpha MEM medium
2.5 ml filtered BSA (694 mg/10 ml in water)
600 ul 1 M HEPES, pH 7.4

2 X FIX BUFFER, pH 7.2

10 mM KCl
274 mM NaCl
8 mM NaHCO3
0.8 mM KH2PO4
2.2 mM Na2HPO4
4 mM MgCl2
10 mM PIPES, pH 7.2
4 mM EGTA

Autoclave, add 11 mM Glucose from a sterile filtered stock and store at 4o C.

2 X TBS, pH 8.0

40 mM Tris 2.42 g
308 mM NaCl 9 g
H20 qsp 500 ml. Adjust pH to 8.0 with NaOH.

MOUNTING MEDIUM (final volume 5 ml)

N-Propyl gallate 6 g/l in glycerol 50% in TBS 1X pH 8.0. Add the N-propyl gallate to 2.5 ml of glycerol the day before and let it dissolve overnight at RT. Add 2.5 ml of 2X TBS on the day of the experiment. 0.02% NaN3 can also be added to the final mix for better preservation.  [If using coverslips instead of dishes, drawing of mounting method here.]

DO CONTROLS EVERY TIME.


Electron Microscopy preparation is different and sample fixations can be found here.

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last revised 16 May 2000 by mc