In Situ Cell Death Detection Kit, TMR red Protocol

Product No. 12156792910

Protocol

Fixation of tissue sections

Roche recommends cross-linking fixatives, such as paraformaldehyde for the TUNEL assay (i.e., when using one of our In Situ Cell Death Detection Kits), to avoid that fragmented DNA of apoptotic cells is washed out of the cells during the assay procedure (this could result in false negatives). In literature, there are also TUNEL references using acetone- or methanol-based fixatives, which do no 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 from time to time until PFA is completely dissolved. Set pH to 7.4 using 5N HCl (approx. 100 μl), and fill to 50 ml final volume with PBS. Prepare this solution fresh; use it after preparation, and discard the what is not used according to local regulations for handling hazardous waste.

Using plastic embedded tissue sections

Plastic embedded tissue sections cannot be used with this kit. The 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. Because UV light also induces DNA stand breaks, terminal transferase could label these stand breaks, resulting in false positive results. For this reason, Roche does not recommend embedding procedures that could induce DNA strand breaks in samples which are to be analyzed using the TUNEL method (i.e., when using one of Roche 's In Situ Cell Death Detection Kits).

Materials

     
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