Immunostaining Protocol

It is time to complete the immunostaining protocol with guidance from Jo-Maree. I must admit that with holidays looming, my note taking was a bit sketchy. I will need to follow up with Jo-Maree to record the correct details of DAPI and Fluorescein Phalloidin stain. This will ensure that I know how to prepare (and order) stocks in the future.

I already prepared a couple of wells at different cell concentrations ready for staining.  We had to delay the protocol, so some of the higher concentration wells are likely a bit over-confluent. It will be interesting to see how they look under the fluorescence microscope.

CELL FIXATION: ‘Dirty’ Biolab

Working in Fume HoodWorking with 4% PFA in Fume Hood

Prior to imaging, I fixed the cells in 4% PFA:

  1. Remove culture media (discard in waste container with bleach)
  2. Wash cells with PBS (discard in waste container with bleach) x 2
  3. Move cells to fume hood
  4. Add 4% PFA to each well for 15 – 20min at room temp (in fume hood)
  5. Remove 4% PFA solution (discard in PFA waste container in fume hood)
  6. Add PBS (make sure cells are covered or they dry out and produce poor images

IMMUNOSTAINING: At lab bench area

Lab BenchWorking at lab bench in the Stroke Group area

  1. Remove PBS from each well
  2. Add 1mL 0.3% Triton X-100 (a strong detergent) to permeabilize cells (make cells permeable – this allows the phalloidin stain to enter the cell structure) for 10 min
  3. Make up DAPI (5mL PBS Tween and 1ul DAPI) and protect from light with aluminium cover
  4. Remove Triton X-100 and add 1mL DAPI solution to each well and incubate at room temp (with aluminum cover to protect from light) for 5 minutes
    Aluminium Cover
  5. Make up Flouroscein Phalloidin (1mL PBS sand 2μL Flouroscein) and protect from light
  6. Remove and discard DAPI solution
  7. Add 1mL PBS 0.1% Tween to each well for 5 min then discard x 3 (i.e. wash with PBS Tween x 3)
    PBS Tween
  8. Add Phalloidin stain to each well.
  9. Incubate at room temp for 1 – 2 hours
  10. Remove Phalloidin solution
  11. Wash with PBS Tween x 2
  12. Add 1mL PBS in each well
  13. Cover with aluminum foil to protect from light.
  14. Cells are ready for imaging. They can be stored in the fridge (with aluminium foil cover) until ready.


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