Thick Specimen Labelling

Protocol for Thick Specimen Labelling.

  • Fix specimen using 4% paraformaldehyde (the length of time depends upon the thickness of your specimen. For confocal microscopy the upper limit is approx 1mm, or its difficult for the antibodies to penentrate.
  • Wash in PBS + 0.2% Triton-X for 20mins (x3)
  • Block using serum free protein blocker or serum from the same species as the secondary antibody for 30mins.
  • Incubate with primary antibody for around 4hrs or overnight at 4°C overnight depending on the thickness of your specimen and the quality of the antibody.
  • Wash in PBS + 0.2% Triton-X for 4hrs or overnight depending on tissue thickness, change the wash at least 3 times during this period.
  • Incubate with secondary antibody for 4hrs or overnight in the dark (from this point specimens should be kept in the dark as much as possible.
  • Wash in PBS + 0.2% Triton-X for 4hrs or overnight depending on tissue thickness, change the wash at least 3 times during this period.
  • Dehydrate the specimen
    • 10mins in 25% Methanol
    • 10mins in 50% Methanol
    • 10mins in 75% Methanol
    • 10mins in 100% Methanol
    • Change Methanol and leave for 1 hr in 100% Methanol
  • Mix Benzyl Alcohol:Benzyl Benzoate ((BABB 1:2 ratio) with Methanol 50% in a 1:1 ratio to give 50% BABB: 50% Methanol. CAREFUL WITH THE BABB ITS TOXIC
  • Incubate in 50:50 BABB/Methanol for 15mins
  • Incubate in 100% BABB for 15mins or until specimen has cleared.
  • Fill a 0.7mm depth depression slide with BABB and transfer specimen into slide.
  • Add a coverslip and seal with nail varnish, make sure seal is very good as BABB is very bad for objectives.
  • Store at 4°C
  • Image specimen within 12-24hrs as fluorescent dyes fade after this

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