Hi everybody,
I'm trying to merge two individually successful protocols to detect apoptotic cells (TUNEL) and endothelial cells (isolectin) in wholemounts of the rat retina.
The problem I'm facing is that I can either get the isolectin stain of the TUNEL working, but not both at the same time. I do TUNEL first, and isolectin second, which requires two overnight washes in total. TUNEL is easily visualized immediately after the reaction as expected. But by the time I get the slides under the scope after isolectin, TUNEL fades away and the tissue is so fragile that 50% of samples are unusable.
Some background on the stains:
TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) is a clever biochemical technique that takes advantage of DNA nicks to identify apoptotic cells. It uses terminal deoxynucleotidyl transferase to catalyze the addition of labeled nucleotides to the 3' end of one of the strands bulging out of the nick.
Isolectin is simply a glycoprotein that sticks selectively to endothelial cells. It is notorious for non-specific binding so proper staining requires ~3 hr of washing in PBS, changed 8 times, after an overnight soak in the staining solution.
Here's my TUNEL protocol:
1. Euthanize rats with overdose of pentobarbitol
2. Harvest entire eyeball and immerse in 4% PFA in PBS (no rocker, Room Temp)
3. 5 min in, remove the cornea and the lens, then let the eye "cups" fix for additional 1.25 hr (total fix = 1 hr, 20 min)
4. Isolate the retina and wash off PFA with 2x2min PBS
5. Dehydrate tissue by the following washes: 1x2min 70% ethanol, 2x2min 95% ethanol, 2x2min 100% ethanol, and 2x2min Xylene (no rocker, Room Temp).
6. Leave retinas in xylene to defatten overnight (no rocker, Room Temp)
7. The next day, rehydrate retinas using the following washes: 2x2min 100% ethanol, 2x2min 95% ethanol, 2x2min 100% ethanol, 2x2min PBS. (no rocker, Room Temp).
8. Permeabilize retinas in 0.3% Triton X-100 in PBS for 15 min. (not on rocker, Room Temp).
9. Next, immerse retinas in Proteinase K buffer solution (10mM Tris) for 2 min (no rocker, Room Temp) followed by 30 min digestion in Proteinase K solution (20ug/ml, 10 mM Tris) (no rocker, 37C) to dissolve proteins bound to DNA.
10. Wash off enzyme with PBS.
11. The retinas are now ready for labeling - they've been defattened, permeabilized, and the DNA binding proteins have been digested away. To prep a positive control, one sample is immersed in DNase (2000 U, 50 mM Tris, 10 mM MgCl2, 1mg/ml BSA)
12. Mix 50 ul of the TUNEL enzyme with 450 ul of the labeled nucleotide (Roche In Situ Cell Death Detection, Fluorescein)
13. Transfer retinas to a 96-well plate and add 50ul of the reaction mixture to each well. A negative control is prepared by adding 50ul of dye only. The wells are covered with parafilm to maintain humidity and incubated at 37C for 1 hr (no rocker)
14. 3x2min PBS washes on rocker.
15. Mount on slides with DAPI mounting solution and coverslip. Excitation 450-500 nm / Detection 515-565nm.
Here's my isolectin protocol:
1. Euthanize rats with overdose of pentobarbitol
2. Harvest entire eyeball and immerse in 4% PFA in PBS in a 32-well plate (no rocker, Room Temp)
3. 5 min in, remove the cornea and the lens, then let the eye "cups" fix for additional 1.25 hr (total fix = 1 hr, 20 min)
4. Isolate the retina and put in PBS
5. Immerse retina in 300 uL isolectin solution (0.1 ug/ul isolectin, 1 mM CaCl2) overnight.
6. The next day, remove isolectin with 8 PBS washes over 3 hours.
7. Transfer eyecups to slides and make four incisions, spaced 90 degrees apart, starting at the ora seratta (the edge) and stopping short of the optinc nerve head (the center). Flatten the retina so it looks like a four-petal leaf, add a drop of anti fading reagent, and pop on coverslip.
8. Visualize under fluorescence microscope. Excitation 590 nm / Detection 617 nm.
One option may be to use a TUNEL stain that is visualized with light microscopy and perhaps less prone to fading. Another option may be to reverse the order, doing isolectin first and TUNEL second or even adding the isolectin stain to the xylene overnight bath or after rehydration. I have very limited experience with double stains so any suggestions would be greatly appreciated!
