Martin Humphries
LAB

Protocols - Classic Imunofluorescence protocol

Written by Guillaume Jacquemet


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Prepare fixation solution:


  • - PFA 4%

  • PFA based fixation is a good method to preserve actin stress fibers
    1. Weight 40g of PFA for 1 L of MilliQ water
    2. 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.)

  1. Boil coverslips 3 times in MilliQ water to clean them
  2. Put coverslips into a 24 well plate
  3. Coat your coverslips with FN overnight (or other ligand)
  4. Block your coverslips with a solution of 10 mg.ml of denaturated BSA (dissolved in PBS)


- Prepare cells:


  1. Trypsin and plate cells
  2. Fixe cells using the right fixation solution for 20 min (or overnight at 4C)
  3. Wash 3 times with PBS

At that stage, fixed cells can be stored in PBS at 4°C until needed.


- Stain cells


  1. 3-4 min 0.5% triton (only if intracellular stainning)
  2. Quenching with 0.1M filtrated glycin (at least 20 min)
  3. Blocking step with 3% non denaturated BSA (optionnal, trying without can improve stainning)
  4. 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
  5. 3 washes with PBS (or with PBS + 1% tween)
  6. Secondary antibody diluted in PBS or 3% BSA (1/200), incubation for 45 min at RT; protected from light
  7. 3 washes with PBS, plus one wash with MilliQ water


- Mount coverslips on slides


  1. Clean glass slides with 70% ethanol and let them dry
  2. Put a drop of monting medium on the glass slide (about 20 uL)
  3. Mount the coverslides, cells facing the glass slide, into the monting medium
  4. Let dry overnight in the dark
  5. Store in the dark at 4°C or at -20°C (long term storage)


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