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Author Topic: TUNEL-Biotin-Avidin-HRP Protocol (SG) for Paraffin Sections  (Read 5696 times)

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Offline ihcwor2

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TUNEL-Biotin-Avidin-HRP Protocol (SG) for Paraffin Sections
« on: March 12, 2003, 10:49:18 PM »
TUNEL-Biotin-Avidin-HRP Protocol (SG) for Paraffin Sections


Solutions and Reagents:

A.   10X Phosphate Buffered Saline (PBS):
Na2HPO4  ----------- 10.9 g
NaH2PO4 ------------ 3.2 g
NaCl ------------------ 90 g
Distilled water ----- 1000 ml
Mix to dissolve and adjust pH to 7.4

B.   3% Hydrogen Peroxide:
To prepare, add 10 ml 30% H2O2 to 90ml PBS or methanol.

C.   TdT Buffer (30 mM Tris base, 140 mM sodium cacodylate, 1 mM cobalt chloride):
             Reaction buffer, 5x (Boehringer Mannheim) --- 40 ul
             CoCl2 (25 mM, Boehringer Mannheim) ---------- 8 ul
Distilled water ------------------------------------------ 160 ul

D.   TdT Enzyme Mixture:
             TdT enzyme ---------------------- 2.0 ul (Boehringer Mannheim)
             Biotin-16-dUTP ------------------ 2.0 ul (Boehringer Mannheim)
             TdT buffer ------------------------- 500 ul (Made above)

E.   Stop Wash (300mM NaCl, 30mM Sodium Citrate)
             1.0 M NaCI ------------------------- 30 ml
             1.0 M Sodium citrate ------------- 3 ml
             Distilled water ---------------------- 67 ml

F.   ABC Reagent:
Depending on sensitivity and morphological requirements, there are varieties of ABC kits and reagents can be selected from. The most commonly used one is VECTASTAIN ABC Kit from Vector Laboratories.      

G.   SG Reagent:
       
Procedure:

1.   Deparaffinize sections in xylene for 3x5min.
2.   Hydrate with 100% ethanol for 2x5min.
3.   Hydrate with 95% ethanol for 2x5min.
4.   Rinse in distilled water.

5.   Hydrogen Peroxide: incubate sections in 3% H2O2 in PBS (or methanol) for 10-15 minutes to block endogenous peroxidase activity.
6.   Rinse in PBS for 2x5min.

7.   Incubate sections in TdT buffer for 10 minutes.

8.   Incubate sections in TdT enzyme mixture for 3 hours at 37 C.

9.   Rinse in stop wash solution for 10 minutes.
10.   Rinse in PBS for 3x5min.
                                                                                                                             
11.   ABC: incubate sections with ABC complex or HRP-streptavidin reagent in PBS for 30 minutes at room temperature.
12.   Rinse in PBS for 3x5min.

13.   SG: incubate sections with SG solution for 2-10 minutes.
14.   Rinse in PBS for 3x5min.

15.   Counterstain with methyl green if desire.
16.   Rinse in distilled water 2x5min.
17.   Dehydrate through 95% ethanol for 5min, 100% ethanol for 2x5min.
18.   Clear in xylene for 2x5min.

19.   Coverslip with mounting medium.


Results:

1.   DNA damaged nuclei -------------------------- blue
2.   Normal nuclei ------------------------------------- pink

TUNEL-Biotin-Avidin-HRP Protocol (SG) for Paraffin Sections
« on: March 12, 2003, 10:49:18 PM »