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In Situ Cell Death Detection Kit, POD Protocol

Product No. 11684817910

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 (which could result in false negatives). In the 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 with 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 handling 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 strand breaks, terminal transferase will label these strand breaks resulting in false positive staining. For this reason, 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).

Use on sheep tissue

This product can be used on sheep tissue. The antibody specifically detects fluorescein. It is irrelevant in this respect, that the antibody is produced in sheep.

Double staining

After applying the In Situ Cell Death Detection Kit, POD to stain formalin-fixed paraffin-embedded (FFPE) liver tissue sections, it is also possible to stain another antigen in the sections using a double staining technique (e.g., a different AP/HRP-based staining kit).

Roche recommends first performing the TUNEL reaction of the In Situ Cell Death Detection Kit, POD for FFPE tissue slides, as described in detail in the package insert. Then, wash the slides which had been assayed by TUNEL in PBS, and perform the immunohistochemistry assay, as described in the double-staining kit you are using. If the double-staining kit also uses a POD-conjugated secondary antibody, you must first inactivate the POD conjugated to the anti-fluorescein antibody of the In Situ Cell Death Detection Kit first. This is easily done by treating the slides with 2% H2O2, followed by thorough washing in PBS.

Materials

     
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