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Double immunofluorescent stain

Double immunofluorescence staining for BCL-6 and else.

Any type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved.
Use cytocentrifuged cells or frozen or paraffin dewaxed sections.
Presence of endogenous fluorescent substances may affect your staining.

Note: This protocol uses a twice repeated sequence of primary and secondary layers in order to amplify the signal, as in the PAP / APAAP technique.

Start at this section for unfixed or acetone-fixed specimens.

1- Mark the slide in order to recognize the area to be stained and label the slide.
2- Fix in acetone for 10 min at RT [this tep can be omitted].
3- Blot the slides and let them dry for 10 min - 2 hrs.
4- Fix in 10% formalin for 10 min in a Coplin Jar.
From this point on, don't let dry the slides at any time.
5- Discard the formalin appropriately and quickly wash a few times in distilled water (from the distilled tap is OK).

 Start at this section for dewaxed, antigen retrieved slides

6- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added. 5 minutes / wash.
7- Briefly blot the slides without letting them dry and then apply 3% human serum (health hazard!) or pig serum a s a blocking agent.
8- incubate with the blocking for 10 min.
9- blot the slides without washing and apply a mixture of the primary antibodies, in a moist chamber, at RT for 1-18 hr.
10- Wash twice.
11- apply a mixture of secondary antibodies (see example below) for 45 min at RT.
12- Wash twice.
13- add the mixture of primary antibodies and incubate for 15 min.
14- Wash twice .
15-  add the mixture of secondary antibodies and incubate for 15 min.

 proceed with steps 16 through 19 if one of the secondary is biotin-conjugated. Else go to 19.

16- dilute the avidin-fluorochrome in TBS/Tween and centrifuge for 15 min.
17-  Wash thrice.
18-  Incubate with the avidin for 15 min.
19-  Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20 and mount.


Example:  Pirmary antibodies: rabbit anti-BCL-6 0.5µg/ml in PBS/BSA/Azide +  mouse anti HA supn 1:100.
Secondary antibodies: biotin-conjugated goat anti rabbit (1:200) + TRITC conjugated goat anti-mouse (1:200).  Alternatively, biotin-conjugated  goat anti mouse (1:200) + goat anti rabbit TRITC (1:200)
Avidin FITC: 1:300

To see the examples click on the thumbnail.
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Blimp1 on skin