Protocols for immunofluorescence

Experimental protocols

Preparation of culture plates

Prior to this laboratory session cells have been seeded into 6 well culture plates with poly-L-lysine coated coverslips on the bottom of each well. The cells have been allowed to attach to the coverslips and grow overnight. The culture medium was removed from each well and the cells were washed three times by replacing the medium with phosphate buffer saline (PBS - a solution which does not put the cells under osmotic stress) and removing. The cells were then fixed in a solution of 4% paraformaldehyde in PBS for 20 minutes, before being washed three times again in PBS.

You will be working in pairs. Three pairs will work with one culture plate, allowing for two coverslips per pair.

Blocking and permeablisation

• Add 3mL of Block / Permeablisation solution (1X PBS containing 1% BSA and 0.1% Triton X100) to each of the remaining wells

• Incubate the coverslips at room temperature for 15 minutes

Treatment with antibodies

The primary antibodies need to be diluted first in Blocking Solution (1XPBS containing 1% BSA). Prepare the following antibody dilutions (make up one tube with both antibodies) :

In order to account for background fluorescence, a negative control should be prepared for the primary antibodies. This coverslip should be placed on a drop of blocking solution rather than diluted antibody.

• Set up 50µL spots of diluted primary antibody on a piece of Parafilm, one for each coverslip, as well as a 50µL drop of blocking solution for the negative control

• Place each coverslip CELL SIDE DOWN on separate spots on the Parafilm

• Incubate the coverslips under a foil cover for 1 hour at room temperature

• Set up three 100µL spots of PBS for each coverslip on a separate piece of Parafilm

• Wash the coverslips by placing each CELL SIDE DOWN on each spot and transferring to the next

• Prepare the secondary antibody solutions by diluting them in Blocking Solution (PBS containing 1% BSA) as indicated below (Make up one tube containing both antibodies) :

• Set up 50µL spots of the secondary antibody solutions on Parafilm and place each coverslip CELL SIDE DOWN on separate spots. In this case, the negative control coverslip should be placed on a spot of diluted secondary antibodies

• Incubate the coverslips under aluminium foil for one hour at room temperature

• Wash the coverslips three times in PBS as before

• Rinse off the PBS by dipping the coverslip in water

• Prepare 10µL drops of Mowiol mounting medium on microscope slides

• Mount the coverslips CELL SIDE DOWN on the drops of Mowiol

• Place the slides under a stream of hot air from a floor heater for 15 minutes.

Imaging using epifluorescence microscopy

Observe the coverslips using the fluorescence microscope as directed by you tutor. Most of the cells visible should be in interphase, allowing you to see a normal nucleus and distribution of tubulin and mitochondria. Due to the poly-L-lysine coating on the microscope, you should see some cells at different stages of mitosis. The three components visualized should be :

• The Nucleus / Chromosomes – The nucleus will appear as a pale green blob with a distinct boundary around its outside. Inside the nucleus should be some unstained regions which resemble bubbles – these are the nucleoli and represent centres of RNA synthesis. During mitosis, the chromosomes condense and the green signal will become stronger. In prophase and prometaphase, you will see the DNA clump together in bright green bundles (the chromosomes) while the clear boundary around the outside of the nucleus will disappear. In cells in metaphase, the chromosomes line up as a line across the middle of the mitotic spindle (the “metaphase plate”), while in anaphase and telophase, you should see the chromosomes separate out into separate bundles at opposite ends of the cell.

• Tubulin – tubulin should appear in interphase cells as a diffuse fibrous red material. In mitosis, from the end of prometaphase, this consolidates into the mitotic spindle – a birdcage like structure with strands of tubulin radiating out from the ends. The mitotic spindle should start to break down by the end of telophase.

• Complex IV / Mitochondria – mitochondria should as tiny blue specks throughout the cytoplasm of the cell – they are actually much smaller than how they are portrayed in diagrams in textbooks.

Things to look out for

• The Stages of Mitosis – see if you can get an image of cells at each stage of mitosis

• Aberrant Cells – HeLa cells have been continuously subcultured since 1952. This means that they have accumulated a lot of mutations, on top of those that led to the cells turning cancerous in the first place. Observe the diversity of cell shape and size in the cells present, along with other anomalies such as cells with more than one nucleus.

• Aberrant Mitosis – some of the mutations HeLas have accumulated include changes to the normal process of mitosis. A more common anomaly is cells dividing in three rather than two – these cells have a three-pronged metaphase plate (like the Mercedes-Benz symbol) and in anaphase you will see the chromosomes being pulled in three directions rather than two.

• Where are the Mitochondria ? – what happens to the mitochondria during mitosis ? How are they parceled out between the two daughter cells ?