Prepare all reagents with ultrapure water. Wear gloves and change them often.

Procedure
Deparaffinize the slides with two 5 min. incubations of clean xylene, followed by three washes with absolute ethanol. Then gradually bring to distilled water.
Place the sections in a radiotransparent slide holder (not metal). (WVR Staining Dish, White,Cat 25608-906; WVR Slide Holders, 24-place, Cat 25608-868). A good alternative are those plastic slide holder for shipping slides in the mail: ask your pathologist).
Immerse slides and holder in 1mM EDTA pH 7.5 (from a 100mM stock) in a beaker. [Optional: cover with a piece of Saran wrap in which you made holes]. Put in a microwave oven and bring to a boil at max power (8 min for 800 ml). Let boil for 15 min at a reduced power (Power 3) so that the liquid continue to simmer. Cool at RT for 30 min to one hour.  Transfer to TBS.

1- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added (TBS-T).
2- Briefly blot the slides without letting them dry and then apply 20µl of the FITC-labelled oligonucleotide cocktail (NCL-EBV). Coverslip. Incubate for 2 hrs at 37°C in a humid chamber.
3- Remove the coversip and wash thrice in TBS-T.
4- add the AP conjugated anti FITC secondary antibody (100 - 300 µl; dilution 1:1,000 or 2,000) and incubate for 1 hr.
5- Wash in TBS-T over 30 min with at least four washes.
6- develop with freshly prepared NBT/BCIP (Roche, 1-681-451 or tablets 1-697-471), by covering the section with 100-300 µl of the solution (filtered with a 45µm filter). Your stain should appear after the first 30 min. and you may develop for 24 hours or more, if needed. You may interrupt the staining by washing the slides in TBS-T and storing them overnight at +4C°, resuming staining the next day. Keep the solution in the dark. You don’t need levamisole, because endogenous AP has been inactivated by high heath.

Post-transplant human tonsil: EBER (NBT/BCIP, blue), CD138 (Fast Red, red) 40x.