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PRETREATMENT
Cut sections at 2-4µm, use a clean water bath with
distilled water, and let the sections dry upright in order to facilitate
adhesion between the section and the charged glass surface. (Slides: Fisher
Superfrost plus cat 12-550-15).
[Optional: put one teaspoon Elmers'glue per waterbath.]Deparaffinize the slides with two 5 min. incubations of clean xylene, followed by three washes with absolute ethanol. Then gradually bring to distilled water.
NOTE: thick sections cause diffusion of the NBT/BCIP precipitate and false negative results.
[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.
PROCEDURE
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 egg white in PBS-BSA-NaN3 as a blocking agent (one egg white in 100 ml PBS + NaN3) (Miller RT et al, Appl Immunohistochemistry 5(1): 63-66, 1997). Incubate for 10 min. minimum. (Use the yolk as directed in Cucinait.com)
NOTE: this passage may be omitted, however subsequent double staining with a biotin based method may need endogenous biotin blocking.3- Wash once in TBS-T.
4- Incubate with skim milk 5% in TBS-T for 10-30 min. (Miller RT et al, Appl. Immunohist & Molecular Morphology, 71(1): 63-65, 1999)
NOTE: skim milk may contain weak proteases which will be useful to further unmask antigens, but may reduce immunostaining by acting on primary Abs.5- Wash once in TBS-T (residual milk can contribute to blocking).
6- blot the slides and apply the primary antibody (100-300µl), in a moist chamber, at RT for 2-18 hr (try first overnight).
NOTE: The moist chamber (see above) must fulfill the following criteria: a) be absolutely flat, b) maintain moisture over 3 days without drying. c) be washable.7- Wash in TBS-T over 30 min with at least four washes. Washing in distilled water may reduce specific and background staining.
NOTE: primary antibody dilution in this system range from 1 µg/ml to 0.01 µg/ml. Above this range you may expect high background. Below this your staining may be erratic. Monoclonal antibodies have a narrower range of dilutions that polyclonals. Typical initial screening dilutions for polyclonal sera are 1:1,000, 1:10,000, 1:30,000.
8- add the AP conjugated secondary antibody (100 - 300 µl) and incubate for 2 hrs.
NOTE: amplification of the system with little increase in background can be obtained as follows:9- Wash in TBS-T over 30 min with at least four washes. Washing in distilled water may reduce specific and background staining. Tap water may kill your AP via fluorides.
Low level: counterstain with Goat anti mouse F(ab) FITC or Goat anti rabbit F(ab) FITC (Jackson Immunoresearch Labs 115-097-003 and 111-097-003) 1:2000/3000 followed by (see below the method) Goat anti-FITC-AP 1:2000 .
High level: counterstain with Goat anti mouse F(ab) FITC or Goat anti rabbit F(ab) FITC 1:3000 (Jackson Immunoresearch Labs 115-097-003 and 111-097-003) followed (see below the method) by Goat anti-FITC-HRP 1:100 (Roche 1426346), followed by FITC-Tyramide (Perkin-Elmer / NEN Cat. # NEL741) and Goat anti-FITC-AP 1:2000.
NOTE: secondary antibody dilution in this system range from 1:500 to 1:2,000.
NOTE: dilute secondary antibodies in TBS-BSA-NaN3. Add to the secondary abs 10% egg white from the above preparation and 10% skim milk (final 0.5%), previously centrifuged to get rid of the particulate suspension. You can also add 5% human or mouse serum ,depending on the tissue target and on the pre-absorption of your secondary Ab.
10- 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 (optional: 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.
Double AP staining:
To perform double staining inactivate the first AP by
bringing the EDTA buffer to vigorous boil, placing the slides in it and
let cool. Then add a second combination of non-crossreacting primary and
secondary Abs. Note that NBT/BCIP is the weakest blocking development and
allows color mixtures more than other combinations.
For color contrast perform a second immunostaining with an
avidin-biotin method, with or without tyramide amplification, and AEC (aminoethylcarbazole) development (see : indirect IHC with AEChere).
Alternatively, use FastRed or NewFuchsin. However we noticed progressive degradation of the FastRed deposition and eventually loss of the red staining over weeks, possibly due to the catalytic activity of NBT/BCIP.
There is no such sensitive development for AP
as NBT/BCIP. You may use INT/BCIP (Roche, 1-681-460), however it changes
to crystals upon storage.
Incubation the sections in absolute methanol turns the
color to bright blue.
Mounting NBT/BCIP slides in xylene gives unpredictable
crystal formation.
