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Double Immunofluorescence Staining for Bcl-6 and Else
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Double Immunofluorescence Staining for Bcl-6 and Else

Dr. Giorgio Gattoretti

Associate Professor of Clinical Pathology
Institute for Cancer Genetics, Columbia University
New York, USA

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 step 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.


Primary antibodies: rabbit anti-BCL-6 at 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

Double IF staining: BCL6 and Ki-67 (MIB 1)

Human tonsil, formalin fixed, paraffin embedded: Rabbit anti BCL-6 (TRITC, red) Mouse anti Ki-67 MIB 1 (FITC green) 40x.

Human tonsil, squamous epithelium (formalin fixed, paraffin embedded section) 40x

 Mouse IgG2a anti Blimp-1 (anti mouse IgG2a, AMCA avidin),  Mouse anti Ki-67 MIB 1 IgG1 (anti mouse IgG1-TRITC), Rabbit anti BCL-6 (anti rabbit FITC).

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