TUNEL Labeling for 50 mm Floating Sections of Rat Brain

This TUNEL procedure can be used in conjunction with Fluoro-Jade staining.  For the most part this procedure follows the Trevigen TACS 2 TdT-Fluor In Situ Apoptosis Detection staining kit (Trevigen 1-800-TREVIGEN, if you order this kit, please tell them Tom Hallam referred you).  The major modification of Trevigen’s procedure is the replacement of the strepavidin-fluorescein that comes with this kit with the strepavidin Alexa-594 (from Molecular Probes 1-800-438-2209).  There is a small amount of excitation and emission of the Fluoro-Jade compound in the rhodamine spectrum, however the Alexa-594 is so bright in comparison to low-level overlap in  fluorescence, the TUNEL positive cells can easily be discerned.  It may be possible to use a strep-Cy5 that would fluoresce in the DAPI spectrum which Fluoro-Jade does not overlap with, but I have not tried this.

Note: if you are NOT looking to perform this procedure with Fluoro-Jade, Trevigen’s strep-flourescein or any of their other markers may work well.  However, I have not tried them.

1) Hydrate free floating sections in 0.1 M PBS  (sections should already be in this)

2) Permeablize Cells

 CytoPore  (do not dilute) Overnight at 4oC in Fridge, (this can probably be cut down to 1 hr)

For this and all of the following steps, you only need to barely cover the tissue sections (usually about 200-300 ml).  But make certain that there are no folds in the tissue, or the staining will be very uneven, or worse yet, some regions of the tissue won’t stain.  I satin 1 section per well in untreated falcon 24 well plates.  I use a glass pipette which has had the tip blunted and bent at a 90 degree angle with a Bunsen burner to transfer the tissue from solution to solution.  During all incubations and washes the tissue is moderately shaken (150-300 RPM).

3) Wash 3X diH2O.

4) Dilute (10X) Labeling Buffer to 1X.  Make sure you have enough for both steps 5 and 6.

5) Put tissue in (1X) Labeling Buffer for 5 min

6) Make Labeling Mixture

     1X Labeling Buffer (step 4)                        100 ml
     Cation:  Mn+                                              2ml / 100 ml
     1X Nucleotide buffer (TdT dNTP mix)      2ml / 100ml
     TdT ENZYME  (Make sure to add last!)     2ml / 100ml

7) Add tissue to Labeling Mixture:  75 min

8) Dilute 10X Stop Buffer to 1X Stop Buffer

9) After reaction is over, add tissue to STOP BUFFER for 5 minutes

10) Wash 3X 0.1 M PBS

******  From this step on, perform as much of the protocol as possible away from light.

11) Make Secondary Labeling Solution with strep-Alexa 594 or another strep-conjugated secondary

 0.5% NGS in 0.1 M PBS with Alexa 594 at 1:200.

12) Wash 3X  0.1M PBS

13) Stain with Fluoro-Jade or different second label

14) Mount and coverslip with DPX (Electron Microscopy Sciences, Inc)

Hints to protect Alexa-594 from photo-bleaching.  Mix the solutions in dim light.  When the slides are staining, wrap the staining tray in aluminum foil.  When necessary, briefly bring the slides out into the light for a few steps and then quickly put them back into a empty drawer or use another method to protect them from light.

LINK TO FLUORO-JADE STAINING PROTOCOL