Immunofluorescence protocols

Most immunofluorescence protocols involve fixation of cells at a specific time point of activation to preserve the distribution of intracellular proteins, followed by cell permeabilization to allow the entry of the antibody into the cell. The primary antibody can either be directly tagged with a fluorophor or, in the indirect approach, a secondary anti-lgC or anti-lgM antibody is tagged. The direct approach is simpler and less prone to background problems. In contrast, the indirect approach offers the advantage of widely available labeled antibodies and the increased sensitivity that results from amplification of the signal (for more details, see Harlow and Lane, 1988). 

Fig. 2 Indirect immunofluorescence protocol. The steps are numbered consecutively.

After 9 days in culture, wash cells twice with phosphate-buffered sahne (PBS, pH 7.4) to remove media and cell detritus. This immunofluorescence protocol is similar to many protocols used extensively in the scientific community, and variations would likely not affect the ability to analyze changes in neuronal morphology. 

Basic protocol for double immunofluorescence staining using primary antibodies raised in two different host species... 

In spite of the promising results obtained by using TIQDT, we found also some problems to be fixed. The main detected problems were (1) the variability in the size and number of thyroid follicles in each animal, (2) the fact that some TGFDs could impair the size or the number of thyroid follicles, but not the concentration/API of T4 signal inside of the quantified follicles, (3) some autofluorescence may be found with the set of immunofluorescence filters used, and (4) clear signs of systemic toxicity were found in eleutheroembryos exposed to some chemicals at the maximum tolerated concentration. For an optimized TIQDT protocol, we increased the... 

The methods outlined below include protocols for direct and indirect immunofluorescence staining, that can be adapted easily for the cell type of interest as indicated in the relevant notes. The principal approaches to flow cytometric analysis, standardization and calibration are then given, followed by two more detailed protocols illustrating quantitation using direct immunofluorescence, and a competitive binding assay, which demonstrates the application of linear amplification of fluorescence. 

Successful cell surface display of the protein can be verified by inducing gene expression via addition of anhydrotetracycline to the culture medium and immunofluorescence staining of the cells using an antibody directed against the protein to be displayed (Protocol 1). Analysis can be performed by fluorescence microscopy or flow cytometry. 

Normal semm is used for blocking non-specific binding sites before adding primary antibodies in immunofluorescence staining protocols. Ideally, it should be of the same species as the secondary antibodies, but if good quality highly cross-absorbed antibodies are used, any normal semm will suffice. 

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. 

This protocol was developed for yeast telomere hybridization experiments, in which we wished to localize both telomeric repeats and either RAPl protein or nuclear pore proteins. For such it has been most successful to fix the cells lightly, remove the cell wall, and carry out the immunofluorescence steps first. Following antibody staining, the cells can be postfixed and hybridized without loss of the immunofluorescence signal. The primary fixation method is based on the work done by Davis and colleagues (Davis and Fink, 1990 Loeb et al., 1993) to immunolocalize nuclear pore components in Saccharomyces cerevisiae. 

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). 

Fig. 2. Treatment of cells with CT976 inhibits ER export of VSV-G, which accumulates in COPII buds at ERES. Cells were infected with ts045 VSV at 40° to accumulate VSV-G in the ER, subjected to various chase protocols, and then stained by double-immunofluorescence for VSV-G (left panels) and the Golgi marker a-mannosidase II (Man II) (right panes). (A, B) No chase (C, D) Cells shifted to 32° for 15 min (E, F) Cells shifted to 32° in the presence of CT976 (G, H) Cells shifted to 32° in the presence of CT976 for 15 min and then washed free of the drug for an additional 15 min. Arrows in E indicate accumulation of VSV-G in foci corresponding to ERESs.

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