Immunofluorescence microscopy staining

Fig. 4 Immunofluorescence microscopy of Giardia intestinalis showing segregation of mi-tosomes during mitosis. A Interphase, prophase, and C telophase cells. Nuclei were stained by DAPI (blue), mitosomes were detected by an antibody raised against GiiscU (red), and axonemes were visualized by the antibody AXO 49 recognizing polyglycylated carboxy-terminal peptides of a- and fl-tubulin (green). Note the proximity of mitosomes (white arrows) to axonemes...

Antineutrophil cytoplasmic antibodies (ANCA) are a class of autoantibodies with varied specificities against particular proteins in the cytoplasmic granules of neutrophils and the lysosomes of monocytes. Indirect immunofluorescence microscopy and enzyme immunoassay have defined two types of ANCA patterns one causing cytoplasmic staining (C-ANCA) the other perinuclear staining (P-ANCA) [346]. Greater than 95% of C-ANCA antibiodies are anti-proteinase 3 antibodies (PR3-ANCA) and greater than 95% of P-ANCA antibiodies are anti-myeloperoxidase antibodies (MPO-ANCA) [347]. 

In immunofluorescence microscopy, specific proteins and organelles in fixed cells are stained with fluorescence-labeled monoclonal antibodies. Multiple proteins can be localized in the same sample by staining with antibodies labeled with different fluorochromes. 

The fate of other nuclear components can concomitantly be analyzed using double-label immunofluorescence microscopy. Cells were fixed and air dried (see above), then incubated with antiguinea pig-specific secondary antibodies conjugated to FITC (10 min), and afterward washed in PBS (10 min). In a second step, the cover slips were incubated for 20 min with an antibody specific for a different nuclear protein (for example, a mouse monoclonal antibody against nucleolar protein fibrillarin). After washing in PBS (10 min), the cells were incubated with an appropriate secondary antibodies (in this case, antimouse-speciflc antibodies) conjugated to Texas Red (Dianova) for 10 min. For the visualization of chromatin, specimens were stained with the DNA-specific fluo-rochrome Hoechst 33258 (5 jug/ml in PBS) simultaneously with the secondary antibody. Finally, the cover slips were washed for 10 min in PBS, dehydrated in ethanol, and embedded in Mowiol (Hoechst, Frankfurt, Germany). 

Regardless of which of the above approaches is used to fluorescently stain the cellular antigens under investigation, conjugation of antibodies to fluorescent dyes is required. As this is a critical step for the performance of high quality immunofluorescence microscopy we will discuss in more detail some protocols for the conjugation of antibodies to fluorescent dyes in the following section. 

Since endocytosis ofLDH was confirmed by TEM images (Figure 13.9), forthe next step, its specific endocytic pathway for membrane entry was determined by immunofluorescence and confocal microscopy. Cells were incubated with LDH-FITC, fixed with 3.7% freshly made formaldehyde, and then stained with either anti-clathrin antibody or anti-caveolin-1 antibody both conjugated to the red fluorescent dye Texas Red (TR). The confocal microscopic images showed that green fluorescent... 

Fig. 9.7 Indirect immunofluorescent staining of SWCNTs-treated F1EK293 cells for day 1-5 by fluorescent microscopy (x 630). Green cadherin red collagen blue cellular nucleus staining with DAPI. The expression levels of cadherin and collagen IV in cells decreased gradually as the culture days increased F1EK293 cells gradually detached from the cell populations as the culture days increased (Grunlan et al., 2004. With permission from Elsevier) (See Color Plates)...

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. 

Male Wistar-Kyoto rats weighing 150 g receive either continuous administration of the test drug by an osmotic pump (ALZA Co., Palo Alto, USA) or saline. Twenty-four hours later, the rats are injected with 1 ml of nephrotoxic serum. At 9, 12, and 14 days, urine samples are collected and urinary protein levels are measured using the Lowry method. At 14 days the rats are sacrificed under ether anesthesia, and both kidneys are removed. Portions of these tissues are processed for light microscopy, immunofluorescence staining and immunoperoxidase staining. 

Initially, we cultured the control neurons without cryopreservation. Neurons cultured on a poly-L-lysine-coated 24-well plate were examined by microscopy and immunofluorescence staining using MAP2 (Figures 1,2). Arrows indicate living neurons. 

Exposure of DRG neurons to inhibitors of membrane protein internalization (either bafilomycin A or chloroquine) prevented the loss of Na conductance normally observed after HSV-1 infection (Storey et al 1998). Furthermore, confocal microscopy of DRG neurons stained with a pan Na channel antibody showed that HSV-1 infection resulted in a marked loss of plasma membrane staining and an overall reduction in immunofluorescence throughout the neurons (Fig. 5). These findings are consistent with the hypothesis that HSV-1 induces Na channel internalization. A possible mechanism of channel loss is suggested by experiments on veratridine-induced loss of surface Na channels in neonatal (but not adult) rats CNS neurons where a rapid internalization of Na channels (Tiy2 15 min) occurs after a rise in intracellular Na concentration (Dargent Couraud 1990, Dargent et al 1994). HSV-1 induced loss of Na currents in adult rat DRG neurons showed a similar dependence on extracellular Na and was inhibited when extracellular Na was substituted with choline, which raises the possibility that a rise in internal Na can mediate the HSV-1-induced internalization. 

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