Double immunohistochemistry for BCL6 and PNA for Germinal Center detection

Double immunohistochemistry for BCL6 and PNA for Germinal Center detection.(on fixed, paraffin-embedded sections)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-biotinperoxidase.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 0.1%NaN3 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 ofthe 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).

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 H
2
O
2
30%.Shake well
Filter with a 45µm filter (optional).
Keep away from direct light, use within 5 min.
Double indirect immunohistochemistryfor 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 0.1%NaN
3
and 0.3% H
2
O
2
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 ofthe 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 thoroug