Double immunohistochemistry for BCL6 and PNA for Germinal Center detection.

Cut sections at 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.] Bake at 60° 1 hr.
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/Baxter slide staining holder S7636. 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. 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.

Indirect immunohistochemistry for BCL-6 with avidin-biotin  peroxidase.

1- Wash the antigen-retrieved, cooled slides twice in TBS-T
2- Briefly blot the slides without letting them dry and then apply a blocking solution (see Reagents and Solutions).
3- incubate with the blocking for 10 min. If one of your antibody is biotin-conjugated, you need at this point to do endogenous biotin blocking.
4- blot the slides without washing and apply the primary antibody, in a moist chamber, at RT overnight . Rabbit anti BCL-6 (Santa Cruz Biotechnology, Santa Cruz, CA: Bcl-6 (N-3): sc-858, use at 0.5 µg/ml)
5- Wash twice in TBS-T.
6- add the biotin-conjugated secondary antibody (100 to 200 µl) and incubate for 45 min. The secondary antibody should be absorbed against human and mouse serum; if not add 1 human and 1 mouse serum before use. Use Dako or Vector at 1:200 - 1:300 dilution.
7- Wash twice in TBS-T.
7bis- block endogenous peroxidase by incubating in 0.1NaN3 and 0.3 H2O2 for 30 min. Wash thrice.
8- add the HRP-conjugated avidin (100 to 200 µl, dilution 1:300 -500) (Dako P0364) and incubate for 20 min. Be careful not to dilute the avidin-HRP in biotin-containing or azide-containing medium.
9- Wash thrice in TBS-T.
10- add 50 ml of  the developing solution (see below ). Protect from direct light.
11- after 5 min, check the staining in your positive and negative controls.
12- check the staining at 10-15 min interval.
13- when staining is complete (usually < 1 hr), wash thoroughly in tap water.
14- counterstain and mount in water soluble mounting medium (glycerol gelatin).

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HRP Developing solution:
For 50 ml developing solution add in order:

• Aminoethylcarbazole (20 mg tablets, Sigma A-6926, dissolved in 2.5 ml NN-DM formamide)
• 50 ml acetate buffer pH 5.5 (52.5 ml of 0.1M acetic acid solution + 196.5 ml of a 0.1M Na acetate solution, bring to 500 ml)
• 25 µl H2O2 30.

Shake well
Filter with a 45µm filter (optional).
Keep away from direct light, use within 5 min.

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Double indirect immunohistochemistry  for BCL-6 and Peanut Agglutinin (PNA) (PNA first staining, BCL-6 second staining)

1- Wash the antigen-retrieved, cooled slides twice in TBS-T
2- Briefly blot the slides without letting them dry and then apply 3 human serum as a blocking agent (health hazard!).
3- incubate with the blocking for 10 min. If one of your antibody is biotin-conjugated, you need at this point to do endogenous biotin blocking.
4- blot the slides without washing and apply biotin-conjugated PNA, in a moist chamber, at RT overnight (minimum for 2 hr.). Biotin-conjugated PNA (Sigma, StLouis, MO, use at 1.0 µg/ml)
5- Wash twice in TBS-T.
6- add the biotin-conjugated goat anti PNA antibody (100 to 200 µl; 1:400) and incubate for 45 min.
7- Wash twice in TBS-T.
7bis- block endogenous peroxidase by incubating in 0.1NaN3 and 0.3 H2O2 for 30 min. Wash thrice.
8- add the HRP-conjugated avidin (100 to 200 µl, dilution 1:300 -500) (Dako P0364) and incubate for 20 min. Be careful not to dilute the avidin-HRP in biotin-containing or azide-containing medium.
9- Wash thrice in TBS-T.
10- add 50 ml of  the developing solution (see above ). Protect from direct light.
11- after 5 min, check the staining in your positive and negative controls.
12- check the staining at 10-15 min interval.
13- when staining is complete (usually < 30 min), wash thoroughly in TBS-T. Be careful to keep the signal to background ratio as high as possible, and no background.
14- do not counterstain and keep in TBS-T.
15- blot the slides without washing and apply the primary antibody, in a moist chamber, at RT overnight (minimum for 2 hr.). Rabbit anti BCL-6 (Santa Cruz Biotechnology, Santa Cruz, CA: Bcl-6 (N-3): sc-858, use at 0.5 µg/ml)
16- Wash twice in TBS-T.
17- add the AlkPhos-conjugated secondary antibody (100 to 200 µl; Goat anti rabbit-AP Southern Biotechnology Associates, Birmingham, AL, 1:300) and incubate for 45 min.
18- Wash twice in TBS-T.
19- add the AlkPhos-conjugated tertiary antibody (100 to 200 µl; Rabbit anti goat-AP Southern Biotechnology Associates, Birmingham, AL, 1:300) and incubate for 20 min.
20- Wash thrice in TBS-T.
7- add 50 ml of  the developing solution (see below ). Protect from direct light.
8- after 5 min, check the staining in your positive and negative controls.
9- check the staining at 10-15 min interval.
10- when staining is complete (usually < 1 hr), wash thoroughly in tap water.
11- preferably postfix in formalin for 4-5 hrs before mounting in water soluble mounting medium (glycerol gelatin).

Do not counterstain, unless you can afford a very gentle hematoxilyn hue in the nuclei.

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AP Developing solution:
For 50 ml developing solution add in order:

• 50 ml Tris-Hcl 0.1M pH 9.2 (exact!) (1:10 from a stock solution 1M)
• Levamisole 1mM (12 mg/50 ml) (Sigma L9756)
• 20 mg Naphtol As BI phosphate (Sigma N2250) (stock solution 40 mg/ml in NN-DM formamide, anhydrous, 0.5 ml aliquots kept at -20°C)
• 10 mg Fast Blue BB Diazonium (Sigma F3378)  salt.

Shake well
Filter with a 45µm filter.
Keep away from direct light, use within 5 min.

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