Hi Stefan!

Phalloidin counterstain is perfect for TUNEL staining and you don't need any antigen retrival. I also advice you a slight dye with phalloidin in order to distinguish clearly nuclear staining ;)

Haematoxylin or methyl green

I totally agree with Haematoxylin if you don't need it a fluorescent staining

I would not use haematoxylin or methil green as they stain DNA ( because are basic die). Phalloidin is OK because it stains F actin that is located in cytoplasm.

I would use Gill's haematoxylin. This version of haematoxylin is lighter and doesn't overstain the nucleus.

I would agree with Hazem (if you don't need florescence) and Jessica in case you need florescence.

Use mayers heamatoxyline from ventana

I would use mayers heamatoxyline for the counterstaining. Its mild bluish colour offers a great contrast with TUNNEL results. It is also simple and cheap.

Hemalun counter stainng is the most commonly used for nucleus.

I also think that haematoxylin is the best choice. Not mask TUNNEL results and allows quantification by image analysis.

I have tried light green and Gills hematoxylin and the pathologist preferred the hematoxylin.

Any alum haematoxylin is fine so long as you use a good differentiation step and remove all but the palest stain from the nuclei. People often overstain very badly when using haematoxylin as a counterstain for a DAB reaction, masking the chromagen. Methyl green at pH 4.6 is great for cytoplasmic antigens but not so good if you need to count the nuclei.

That sounds excellent. You can always remove excess stain with acid alcohol if your nuclei are too dark.

use counter cytoplasmic stain EOSIN

We also typical use DAB as the chromogen and light green as the counterstain. I also prefer a lighter hematoylin or light green counterstaih, the dark blue staining of the hematoylin is not as well contrasted against the dark brown staining of the DAB.

I prefer to use Methylgreen, I like more the contrast between green and brown. And remember, when your target staining is in the nucleus is better you use the counterstain for cytoplasm, in this case Methylgreen. In another words, the TUNEL is brown and is localized in the nuclei, then I use Methylgreen for stain the cytoplasm.

You don't specify what chromogen or detection method you are using for your TUNEL...fluorescent, DAB, other? For brightfield DAB imaging, I find Methyl green rather than hematoxylin is more conducive to automated image analysis/nuclei counting, as it doesn't mask DAB signal.

Antigen retrieval is not usually necessary for the counterstains, but vital for the TUNEL stain itself in paraffin sections. Triton is one of many antigen retrieval methods. Proteinase K is another method often upreferred for TUNEL stains, but it requires careful titration for each tissue type and fixation protocol you use, and good use of positive controls.

If you are doing fluorescence staining, your "background" could be due to autofluorescence induced by aldehyde fixatives and natural pigments in the tissue-especially in erythrocytes. These show up worst in the blue-green-yellow range. You may wish to alter your dye choices to the farther red spectrum dyes to avoid this, and use care adjusting your imaging/camera setup. Also, avoid glutaraldehyde in your fixative for fluorescence staining.

Depending on your detection method. If you use DAB, then hematoxylin is the best nuclear stain. You should make sure it is not too dark, leaving your slides in the hematoxylin solution for 1-2 minutes only. If you use the fluorescent detection, use DAPI. In my experience Phalloidin with Texas Red conjugate did not require antigen retreival. Obviously you can only use it if you use the FITC detection.

Good luck and happy new year!

Agnes

Dear Ehrentraut,

We published a paper recently using this method. I send you a link of the manuscript.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492052/

Have a good look!

Best wishes!

Felipe

One of the most widely used methods for detecting DNA damage in paraffin sections is TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining.TUNEL staining was initially described as a method for staining cells that have undergone programmed cell death, or apoptosis, and exhibit the biochemical hallmark of apoptosis.For both immunohistochemical stains, immunoreactive cells were visualized with DAB chromogen (DAKO) followed by a Mayer haematoxylin counterstain

Hi Stefan,

if your chromogen is DAB, you can use both Mayer's haematoxylin or methylene blue.

Haematoxylin stains nuclei, while methylene blue stains both nucley and cytoplasm.

Nevertheless, I prefere not to use counterstain because tunel labels nuclei and with the counterstaining the intensity of labeling will be falsed.

Without counterstaing you can also quantify your labeling using Image J.

Good luck!

Lorena

I prefer light green as nuclei remains unstained and effective result with appreciable contrast is achievable.

I use TUNEL kit (Roche) TMR red (fluorescence detection) and counterstain with Hoechst 33258, stock solution 1mg/ml in distlled water and working solution 0,15ug/ml PBS. Incubation time, 2 min.

We do a lighter hematoxylin as a counterstain. The TUNEL procedure we do includes a Proteinase K step that works as enzyme digestion retrieval .

Agreeing with the methyl green above. This is the standard practice.

Counterstaining with a lighter hematoxylin is a good option.