Double staining for Ag and Ki-67, TUNEL or BCL-6. Cut frozen tissue sections (spleen, lymph nodes) in a cryostat at  ~ 4µm, on plain glass slides (Adhesive -coated slides are sometimes too hydrophobic, but fine if you have them).Let them dry approx 1 hour in a dry atmosphere at RT. Antibodies (anti-Pe) in the sections degrade more rapidly than tissue antigens. Proceed with staining within 24 hours or freeze the slides.

Antigen-specific staining: (Pe staining of Pe-immunized animals).

1- Fix the sections in 10% buffered formalin for 10 min. at RT. 2- Wash briefly in tap water, then once in distilled water and then in TBS 0.05M pH7.5 + 0.01% Tween 20 (TBS-T). 3- Briefly blot the slides without letting them dry and then apply 3% human serum as a blocking agent (health hazard!). 4- incubate with the blocking for 10 min. 5- blot the slides without washing and apply Phycoerythrin (B-Pe or R-Pe) 0.5µg/ml in PBS-BSA-NaN 3 , in a moist chamber, at RT for 1-18 hr. 6- Wash twice in TBS-T. 7- counterstain with rabbit anti B-Pe 1:2000 (0.5 µg/ml Cortex Biochem). You may add 1% mouse serum to the anti-Pe antibodies, unless you plan to use mouse monoclonal further (see below). 8- wash twice in TBS-T. 9- block endogenous peroxidase by incubating in TBS-T 0.1%NaN 3 and 0.3% H 2 O 2 for 30 min.  Wash thrice. Top of Page             Choose next steps: HRP-conjugated secondary Ab Tyramide amplification Biotin-conjugated secondary Ab

HRP-conjugated secondary Ab choice:

10- add the HRP-conjugated Goat anti rabbit (Vector 50 to 100 µl, dilution 1:100) and incubate for 30’.  Swine anti-rabbit (Dako) can be used at 1:300 dilution. If you use this step for tyramide amplification (see below)  dilute the Goat anti Rabbit to 1:200. 11- wash thrice in TBS-T. 12- develop (see protocol ). Protect from direct light. Continue

Tyramide amplification choice:

Use tyramide amplification hereContinue

Biotin-conjugated secondary Ab choice:

10- add the biotin-conjugated goat anti-rabbit (50 to 100 µl, dilution 1:200) and incubate for 30 min. 11- wash thrice in TBS-T.

From this step on follow either "Development (Avidin-HRP) " or "Double staining ".  

Development (Avidin HRP):

12 -add the HRP-conjugated avidin (50 to 100 µl, dilution 1:300 -500) and incubate for 20 min. Be careful not to dilute the avidin in biotin-containing medium. 13- wash thrice in TBS-T. 14- add 50 ml of  the developing solution (see below ). Protect from direct light. 15- after 5 min, check the staining in your positive and negative controls. 16- check the staining until complete, dense staining is obtained, but background is still low. 17- when staining is complete, wash thoroughly in tap water. Counterstain. 18- transfer to TBS-T. Warm. Mount.  

Double staining (BCL-6, TUNEL)

11 -fix in absolute methanol RT for 10 min. Warning: methanol dissolves AEC and blocks HRP. Do this step only after biotin-secondary or tyramide-biotin. 12- rinse in water, bring to TBS-T. 13- add the HRP-conjugated avidin (50 to 100 µl, dilution 1:300 -500) and incubate for 20 min. 14- wash thrice in TBS-T 15- add 50 ml of  the developing solution (see below ). Protect from direct light. 16- after 5 min, check the staining in your positive and negative controls 17- check the staining until complete, dense staining is obtained, but background is still low. 18- rinse in TBS-T         Choose next steps: Double staining with TUNEL Double staining with antibodies  

Double staining (antibodies):

19- apply the diluted antibody (BLC-6, Ki-67, others) 20- wash twice in TBS-T 21- counterstain with an AP-conjugated secondary antibody (Typically SBA goat anti rabbit-AP 1:100, rabbit anti goat-AP 1:300) 22- wash twice 23- [optional] add a tertiary layer (rabbit anti-goat-AP on a goat anti-rabbit-AP) 24- wash thrice 25- develop AP (see protocol). Double staining (TUNEL): 19- warm the section in TBS 20- prepare a warm incubation box 21- set up the TUNEL mixture (see data sheet) 22- incubate 30 min at 37°C. 23- wash thrice in TBS-T 24- apply goat anti-FITC-HRP 1:200 25- wash twice 26- counterstain with rabbit anti-goat-AP 1:200 27- wash thrice 28- develop AP (see protocol) HRP development protocol (AEC): 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 wellFilter with a 45µm filter (optional).Keep away from direct light, use within 5 min.

AP development protocol (Diazonium salts): 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 (stock solution 40 mg/ml in NN-DM formamide, anhydrous, kept at -20°C) (Sigma N2250, the cheapo)
• 10 mg Fast Blue BB Diazonium salt  (Sigma F3378)

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

Golden rules during and after development :

• after 5 min, check the staining in your positive and negative controls.
• check the staining at 10-15 min interval.
• when staining is complete (usually < 1 hr), wash thoroughly in tap water.
• 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.

click on the image to enlarge  

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