Hi all,
I've been trying to troubleshoot using the In Situ Cell Death detection kit from Sigma/Merck with fluorescein for use on PFA-fixed rat brain sections. I'm currently trying the staining on DNAse-treated sections first (the final goal is to combine it with an immunostaining).
I'm getting an enormous amount of nonspecific staining (whole section is bright green, but I also have streaks of staining in the corpus callosum). I have some positive cells, but the background in brain areas I am interested in is so high that the staining is basically not useable at all.
The kit recommends diluting the reagents, but I wonder if I could try to improve on incubation time and maybe some kind of blocking solution as well? Do you have some initial dilutions I could start out with? A magic blocking ingredient?
Thanks,
Susan
My current protocol is like this:
1) mount section, dry, wash with PBS
2) permeabilize for 30min at room temperature in 0.3% Triton-X and 2%BSA
(the recommended 1 min, with 0.1% Triton-X on ice incubation gave me no stained cells at all, so I used my regular permeabilization step for immunostaining instead)
3) wash and incubate with kit (per kit instructions)
4) wash again, counterstain with DAPI
5) wash, coverslip, image
Hi Susan,
TUNEL assay is very sensitive method to determine the DNA fragmentation in cells underwent apoptosis. The exposed 3'-OH of the broken DNA can be catalyzed by Terminal Deoxynucleotidyl Transferase (TdT) with labeled dUTP, which can be detected using TUNEL either by light or fluorescence microscopy depending on the nature of detecting markers (DAB or Florescence).
It seems that you are trying to analyze the DNA fragmentation in PFA-fixed rat brain histological sections. Since you are using DNAse-treated sections, it is quite possible that DNAse treatment might have generated lots of fragmented DNA in the sections on the slide. The increased DNA fragmentation (as positive control) in the section will have high TdT-dUTP labeling, which in turn can generate lots of florescence. Hence, you are getting lots of background florescence (scientific reason).
I would suggest you to directly go for your experimental slides wherein you have taken control as well as treated slides and see whether you are getting similar high background. If yes, then try to fix the followings (technical reasons):
1) Reduce the time of TdT labelling
2) Wash the slides thoroughly with wash buffer (double the washing of the slides)
3) Reduce the Illumination value and exposure time until you get the flourscence from TUNEL positive apoptotic cells with no background illumination.
We have carried out the DNA fragmentation analysis in PFA-fixed rat brain sections, hope the basic methods as well as florescence microscopy protocol. See the link for our published papers that could be useful to you.
1. Generation of hydrogen peroxide mediates hanging death-induced neuronal cell apoptosis in the dentate gyrus of the rat brain. Brain Research Bulletin
Volume 95, June 2013, Pages 54-60.
https://www.sciencedirect.com/science/article/abs/pii/S0361923013000452?via%3Dihub
2. p53 activation and mitochondria-mediated pathway are involved during hanging death-induced neuronal cell apoptosis in dentate gyrus region of the rat brain. SpringerPlus volume 2, Article number: 407 (2013).
Article p53 activation and mitochondria-mediated pathway are involve...
Good luck
Best
1. If you’re using powder BSA and making a solution in triton x-100, try increasing the concentration to a 5% BSA and syringe filter the solution after incubating in warm bath x 30 min. BSA ‘crystals’ that are not fully dissolved- even after lots of vortex it- can be notorious for high background fluorescence.
2. Lower the staining concentration (whatever you’re doing now, dilute 2, 4x).
3. Increase wash times and make more vigorous like on a shaker.
4. Keep slides/experiment in the dark with tinfoil.
5. Wash with 5% filtered BSA/TBST or PBST solution 3x for 5 min each.
6. Include + and - controls to make your optimization shorter/quicker.
7. Wash 3x after DAPI counterstain.
8. Use ProLong Diamond mounting reagent which reduces background signal and cross-channel excitation.
9. If doing imaging in multiple channels, image the channel of interest first then your counterstain to minimize cross-channel excitation and thus high background.
10. Lower your microscope‘s laser power/brightness at the source. Avoid long exposure times which will quickly photobleach your slides. Optimize this in a small section of your slide before imaging the whole thing.
11. Modify things one by one. Start with increased washing and lower stain concentrations, and if applicable use filtered BSA. Normal serum is also an alternative to BSA but more $$.
Hope that helps! Feel free to message if you have any questions.
Hello, could you provide me with more information regarding your technique so that I can help you? Do you perform the technique in frozen sections (Cryostat)? At what concentration do you have the PFA and for how long do you leave it fixing? Or do you have them included in paraffin?
Hi there, DAPI staining is used for living cells and specifically binds to the Adenine and Thymine base locations on the natural DNA, but TUNEL staining is a method for detecting apoptotic cells that have fragmented DNA; this stain is attached to DNA fragment terminals.