Fixation procedures immunofluorescence

We therefore present two different procedures for fixing and preparing yeast samples for FISH. The first of these is suitable for rDNA hybridization and/or tubulin immunofluorescence (preservation of tubulin requires high concentrations of formaldehyde and rapid fixation). The second protocol is designed for hybridization with sequences of lower copy number and r immunofluorescence with antibodies to nuclear proteins such as RAPl or nuclear pore components. These antigens are extremely sensitive to overfixation, and so the cells must be fixed lightly for immunostaining, then postfixed for hybridization. If possible, use the first procedure because it is simpler and more reliable. 

Cells are grown on cover slips. After fixation they are permeabilized with detergent and incubated in a medium that supports run-on replication. Subsequently, cells are fixed and processed for immunofluorescent labeling. The labeling procedure can be completed in one day. 

The immunofluorescent labeling procedure described in this section is analogous to that described in Section III,A. It involves fixation and permeabilization of the cells and denaturation of the DNA in order to give the antibodies access to the halo atoms of the incorporated IdU and CldU nucleotides. A very similar dual-labeling protocol, more specifically for flow cytometry rather than for fluorescence microscopy, has been described by Aten et al. (1994). 

The use of this wax for immunohistochemistry has previously been reported in combination with both formalin fixation (4) and acid-ethanol fixation (5). Its routine use for immunohistochemistry was described in 1991 (6), and it has been used in my laboratory as the method of preference for immunohistochemistry for 11 yr (e.g., 7,8). It can be used for any immunostaining procedure, e.g., immunofluorescence or immunoperoxidase, with or without counterstaining, or combined with In situ hybridization (ISH) (9). 

Fixation by Boiling. The boiling fix method (see Protocol 9.3, Method 4) is often tried when more standard fixations fail. For example, immunofluorescent analysis of a number of centrosome proteins requires this fixation method. This procedure also results in devitellinization of virtually all of the embryos. 

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