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Author Topic: TUNEL-Boitin-Avidin FITC Protocol for Paraffin Sections  (Read 5283 times)

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Offline ihcwor2

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TUNEL-Boitin-Avidin FITC Protocol for Paraffin Sections
« on: March 12, 2003, 10:42:32 PM »
TUNEL-Boitin-Avidin FITC Protocol 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.   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

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

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

E.   Streptavidin-FITC Reagent:
Purchase from Vector Labs and use 1:50 dilution.
     
F.   PI Stock Solution (1mg/ml or 1.5 mM):
             PI (Propidium Iodide) -------- 1 mg
      Distilled water ------------- 1 ml
      Store stock solution at 4 C (or aliquot and store at –20 C), protected from light.  When handled properly, solutions are stable for at least six month.

G.   RNase A Stock Solution (1mg/ml)
      RNase A --------------------- 1 mg
      Distilled water -------------- 1 ml
            Aliquot and store at –20 C freezer

H.   PI Working Solution:
      PI Stock Solution ------------ 2 ul (final dilution 1:1000 or 1ug/ml)
      RNase A Stock Solution --- 20 ul (final dilution 1:100 or 10ug/ml)
      PBS ------------------------------ 2 ml


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.   Rinse in PBS for 2x5min.

6.   Incubate sections in TdT buffer for 10 minutes.

7.   Incubate sections in TdT enzyme mixture for 2 hours at 37 C.

8.   Rinse in stop wash solution for 10 minutes.
9.   Rinse in PBS for 3x5min.
                                                                                                                             
10.   Incubate sections with streptavidin-FITC (1:50, Vector Labs) in PBS for 30 minutes.
11.   Rinse in PBS for 3x5min.

12.   Counterstain with PI Working Solution for 20 minutes at 37 C.
13.   Rinse 3x5min in PBS.

14.   Coverslip with aqueous mounting medium (FluoroMount) and seal with nail polish.

15.   Observation using a fluorescence microscope with appropriate filters. Store slides in the dark at 4 C.


Results:

1.   DNA damaged nuclei ------------------------- green
2.   Normal nuclei ----------------------------------- red

TUNEL-Boitin-Avidin FITC Protocol for Paraffin Sections
« on: March 12, 2003, 10:42:32 PM »