Postfixation of the Immunostaining Signal

Either formaldehyde or EGS can be used as fixing agents in order to prevent signal degradation during the FISH procedure. We usually try formaldehyde first and only test EGS whenever formaldehyde does not work. A critical point is postfixation time. Longer times will, in general, better preserve the immunostaining signal, but they will reduce the efficiency of in situ hybridization. We usually perform postfixation for 15 min at 37°C, as indicated in Subheading 3.3.1., step 2 and Subheading 3.3.2., step 2, but this time can be reduced to 10 min as tested empirically. As stated in Subheading 3.1.3., some epitopes do not need postfixation. In this case, proceed to Subheading 3.4.

3.3.1. Postfixation by Formaldehyde

2. Incubate the chromosomes in 20 |L of 3.7% formaldehyde in PBS under a cover slip for 15 min at 37°C in a humid chamber in the dark (see Note 8).

3. Wash three times in PBS, 5 min each, and proceed to Subheading 3.4.

3.3.2. Postfixation by EGS

2. Incubate the chromosomes in 20 |L of 50 mM EGS solution (freshly made in PBS) under a cover slip for 10-30 min at 37°C in a humid chamber in the dark (see Note 8).

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