Immunofluorescence protocol

  1. Place slides in over for 15-20 mins
  2. Deparafinization: In xylene 3 x 5 mins
  3. Rehydration:
    2 x 10 mins - 100% EtOH
    2 x 10 mins - 95% EtOH
    2 x 10 mins - 70% EtOH
    2 x 5 mins - dH2O (can leave it in)
  4. Antigen retrieval: by boiling the slides in sodium citrate solution for at least 10 mins

Sodium citrate solution:
2.94g sodium citrate + 1L dH2O

  1. Put slides in water to cool down
  2. Arrange slides in the rack w/ their plastic covers
  3. Wash w/ washing buffer 1mL for each slide 3 x 5 mins

1x PBS + 0.1% Triton

  1. Blocking: w/ blocking solution 200µL for each slide for 1 hour at room temperature

Blocking solution:
9mL washing buffer + 500µL FBS + 500µL goat serum

  1. Add 200µL of 1st Ab:
    Prepare the antibody solution 1:1000 ratio** and block overnight in 4°C
  2. Wash w/ washing buffer 1mL 3 x 5 mins (don’t need to change tip)
  3. Add the 2nd Ab 200µL w/ dilution 1:1000 ratio mix well and incubate for at least 1 hour in the dark at room temperature (if >2 hours put into the fridge)
  4. Wash 3 x w/ 1mL washing buffer
  5. Wash w/ 1mL dH2O
  6. Remove the slides from the rack and dry them well w/out touching the tissues
  7. Add small drops of DAPI + cover w/ glass (mounting the slides)
  8. See the results on the immunofluorescence microscope

* blocking buffer can stay in the fridge up to 1 week, throw out if precipitate
**1:500 ratio for CD3

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