Centre Optical Instrumentation Laboratory

Immuno-labelling In Very Thick Specimens

1. Fix specimen using 4% paraformaldehyde (the length of time depends upon the thickness of your specimen. For confocal microscopy the upper limit is approx 2mm, or its difficult for the antibodies to penentrate
2. Wash in PBS + 0.2% Triton-X for 20mins (x3)
3. Block using serum free protein blocker or serum from the same species as the secondary antibody for 30mins
4. 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.
5. 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.
6. 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.
7. 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.
8. Dehydrate the specimen
(i) 10mins in 25% Methanol
(ii) 10mins in 50% Methanol
(iii) 10mins in 75% Methanol
(iv) 10mins in 100% Methanol
(v) Change Methanol and leave for 1 hr in 100% Methanol
9. Mix Benzyl Alcohol:Benzyl Benzoate ((BABB 1:2 ratio) with Methanol 50% in a 1:1 ratio to give 50% BABB: 50% Methanol
10. Incubate in 50:50 BABB/Methanol for 15mins
11. Incubate in 100% BABB for 15mins or until specimen has cleared.
12. Fill a 0.7mm depth depression slide with BABB and transfer specimen into slide
13. Add a coverslip and seal with nail varnish, make sure seal is very good as BABB is very bad for objectives.
14. Store at 4°C
15. Image specimen within 12-24hrs as fluorescent dyes fade after this