In Situ Cell Death Detection Kit, Fluorescein方法和问题

Protocol 实验方法

Measurement of stored cryopreserved tissue 测定冰冻组织样品。

Dehydrated fixed tissue sections (2 min dehydration in absolute ethanol, storage at -15 to -25 °C) can be placed directly into the permeabilization solution. They can also first be rehydrated in PBS.

Double labeling with propidium iodide 使用双染 PI 无法显色

Double labeling is not possible because fluorescein fluorescence is quenched. 双染对于 Tunel 问题很难实验，问题在于 fluorescein 荧光很容易淬灭。Counterstaining is only possible successively (first apoptosis determination, then propidium iodide staining). For additional guidelines, consider the following:

先进行 Tunel 染色，再染 PI.

当然必须需要双染可以采取以下 2 种方法：

• Use only 0.5 μg/ml PI as DNA stain 使用 0.5ug/ml PI 染核
• Take TO-PRO-3 instead of PI. 使用 TO-PRO- 3 替代 PI

Fixation of tissue sections

Roche recommends cross-linking fixatives, such as paraformaldehyde for the TUNEL assay (i.e., when using one of our In SituCell Death Detection Kits) to avoid that fragmented DNA of apoptotic cells is washed out of the cells during the assay procedure (which could result in false negatives). In the literature, there are also TUNEL references using acetone- or methanol-based fixatives which do not cross-link cellular components. When using such fixatives it is possible that apoptosis is underestimated due to a loss of fragmented DNA. Fixation of cultured cells wih the formalin-free Accustain^® (Sigma) is as good as freshly prepared paraformaldehyde, but faster (fixation time is only 15 min compared to 60 min with paraformaldehyde). Alternatively, fixation with 2% glutaraldehyde can also be tested.

Preparation of 4% Paraformaldehyde (PFA)

In a screwcap bottle, mix 2 g PFA, 40 ml PBS and 100 μl 5N NaOH. Incubate at +56 °C in a water bath; shake periodically until PFA is completely dissolved. Set pH to 7.4 using 5N HCl (approx. 100 μl), and fill up to 50 ml final volume with PBS. Prepare this solution fresh, use it just after preparation, and discard the rest according to local regulations for hazardous materials.

Using plastic embedded tissue sections

Plastic embedded tissue sections cannot be used with this kit. Polymerization of plastics used for tissue embedding (e.g., methyl methacrylate, glycol methacrylate, or epoxys like araldite and epon) is often induced by UV light. UV light also induces DNA stand breaks, and terminal transferase will label these strand breaks, resulting in false positive staining. Roche does not recommend any embedding procedures which could induce DNA strand breaks for samples which are to be analyzed by the TUNEL method (i.e.,when using one of our In Situ Cell Death Detection Kits).

Troubleshooting

Reducing background when using In Situ Cell Death Detection Kits

The best approach to reduce non-specific background depends on the results obtained using controls. When cells are incubated with fluorescein-dUTP, but without terminal transferase are false positive, try to wash the cells thoroughly, reduce the concentration of fluorescein-dUTP, and, if possible, consider using an alternative permeabilization procedure.

If false positives are produced only in reactions which include both fluorescein-dUTP and terminal transferase, the best way to reduce false positives is a reduction in enzyme concentration or a change in the permeabilization procedure.