Use flat-bottom 96 well microplates.
Use TBS instead of PBS for all the washings but
the first.
Perform all steps at +4°C, up to the fixation point.
1- Resuspend the cells in complete medium at a convenient concentration (each microtiter well contains 300 µl and you may not stain less than 250.000 cells per well).
2- Place 0.25 to 3 millions cell per well of a flat bottom 96 wells plate.
3- Centrifuge at 1000 rpm for 5 min with a slightly tilted plate, so that the cell will collect at one side of the well.
4- Aspirate the supernatant from the opposite side.
5- Vortex the whole plate in order to resuspend the cells in the few µl left.
6- Fix for 10 min in 200µl lysing buffer 1x (made from the 10x stock solution). Several companies sell this lysis buffer. Alternatively see J Immunol Methods 124: 103, 1989. Plain formalin or gluteraldehyde fixation makes the cells very sticky.
7- repeat steps 3 to 5
8- add 250 µl of PBS to each well
9- repeat steps 3 to 5 (centrifuge, discard the supn and vortex)
10- add 50µl of goat serum (1:10 in TBS) to each well and leave for 15-45 min. Alternative blocking sera may be used (e.g. human, providing that the same serum is added to the secondary antibody used). Blocking with milk should be avoided because antigenicity may be reduced.
11- fill up with TBS
12- repeat steps 3 to 5 (centrifuge, discard the supn and vortex)
13- add the diluted antibodies (50 to 100 µl/well) and incubate for 45 min to overnight. Do not exceed 24 hrs of incubation because the formalin bonds begin to become loose thereafter. Anti BCL-6 antibody PGB6 may be used 1:10 from the supernatant. Do not use anti BCL-6 polyclonal antibodies because the results may be unpredictable.
14- repeat steps 3 to 5 (centrifuge, discard the supn and vortex)
15- add 250 µl of TBS to each well
16- repeat steps 14 and 15
17- add the secondary antibody (50 to 100 µl/well) and incubate for 45 min. Do not exceed 24 hrs of incubation because the formalin bonds begin to become loose thereafter. SBA Goat anti mouse AP and Gat anti Rabbit AP can be used 1:200 in TBS-BSA NaN3.
18- repeat steps 14 and 15 and 3 to 5 (centrifuge, discard the supn and vortex) twice at 5-10 min interval at least.
19- add 50 µl of the developing solution, centrifuge shortly (1 min) to settle the cell to the bottom. Protect from direct light.
20- after 5 min, check the staining in your positive and negative controls.
21- check the staining at 10-15 min interval.
22- when staining is complete (usually < 1 hr), add 100µl distilled water, followed by 100µl 10% formalin.
23- repeat steps 3 to 5 (centrifuge, discard the supn and vortex),
24- add 25µl of 10% formalin, heat at 37°C while you warm the gelatin under the running hot water (do not use the MWO).
25- add 1-2 drops of bubble-free melted gelatin to each well, shake gently, quickly centrifuge the plate and cool it. Seal the wells with a film of parafilm.
Developing solution:
For 50 ml developing solution add in order: