Immunofluorescence staining for

Figure 26.5 Immunofluorescent staining for transformed airway epithelial cells with antibodies specific for (A) keratin 18 (a marker of epithelial cells) and (B) ZO-1 (marker of tight junction formation). Both markers indicate that the cells have retained epithelial characteristics after transformation. The staining for the presence of ZO-1 at the periphery of the cells indicates that the cells have not lost their polarity and can form tight monolayers that will generate a transepithelial resistance. This is a particularly attractive feature for the analysis of ion transport and transcellular transport of macromolecules. ZO-1 is also found in the nucleus and can also be detected by the anti-ZO-1 antibody.

Fig. 2 EGCG inhibits FAK activity. (A) Cells were plated in the presence of various concentrations of EGCG for 12 hours. Total cell lysates were subjected to western blot analysis. (B) Immunofluorescence staining for p-FAK (green) in BxPC-3 cells in the absence or presence of 100 pM EGCG. Staining with TOPRO-3 (red) visualizes nuclei...

Long DJ, Buggs C. Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues. I. Mol. Histol. 2008 39 1-4. 

Beutner, E.H. (1971) Defined immunofluorescent staining past progress, present status, and future prospects for defined conjugates. Ann. NY Acad. Sci. 177, 506-526. 

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

As stated above, this approach is applicable only when primary antibodies are raised in different species. For double or multiple indirect immunofluorescence staining with primary antibodies raised in the same species, see Sects. 8.2. and 8.3 below. 

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)...

Figure 25. A-D Immunofluorescence staining of vmculrn in vascular smooth muscle cells on day 3 after seeding on polymeric surfaces (medium supplemented with 10% fetal bovine serum). A poly(DL-lactic acid), PDLLA B block copolymer of poly(DL-lactic acid) and poly (ethylene oxide) (PEO), PDLLA-6-PEO C, E PDLLA-6-PEO with 5% GRGDSG-PEO-6-PDLLA D, F PDLLA-6-PEO with 20% GRGDSG-PEO-6-PDLLA. E, F Immunoperoxidase staining of bromodeoxyuridine (arrows) incorporated into DNA newly synthesized in vascular smooth muscle cells cultured for 3 days in serum-free medium on PDLLA-Z)-PEO with 5% (E) or 20% (F) GRGDSG-PEO-6-PDLLA. Cells counterstained with light green. Bar=100 pm [41].

Use the relevant visualization steps described in Subheading 3.3. for IGSS, IPO, or lAP, in sequential reactions. Sections stained for immunofluorescence should be mounted with a glycerol-gel solution containing an antifading agent such as Vectashield (Vector). 

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. 

Frozen sections, as well as sections of formaldehyde-fixed and paraffin-embedded tissues, can be used for direct or indirect immunofluorescence staining after antigen retrieval using microwave heating. Although frozen sections yield higher sensitivity than that obtained with paraffin sections, the latter approach is necessary for retrospective studies of archival specimens. Moreover, fresh tissue is not always available. 

Figure 2. Cultured pulmonary artery endothelial cells stained for tubulin (red), actin (green) and DNA (blue). The dual immunofluorescence procedure used rabbit anti-actin IgG and mouse anti-alpha tubulin IgG as primary antibodies. The secondary antibodies used were Texas Red-conjugated goat, anti-rabbit IgG and FITC-conjugated goat, anti-mouse IgG. The sample was also stained with the DNA-specific dye Hoechst 33342. Scale bar is equal to 20 microns.

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. 

For cells expressing GFP fusion protein, Hoechst 33342 or Draq5 nuclear stain is added to the fixative to ready the cells for image acquisition. For immunofluorescence staining, the cells are incubated with PBS-TB (PBS with 0.2% Triton X-100, 0.1% bovine serum albumin [BSA]) for 10 min to permeabilize the cell membranes. Primary antibody is then added at 0.5 to 5 pg/mL in PBS-TB and incubated at room temperature for 1 hr or at 4°C overnight. The wells are then washed two times with PBS-TB. Fluorescently labeled secondary antibody is added at 2 pg/mL together with 10 pg/mL Hoechst 33342 nuclear stain in PBS-TB and incubated at room temperature for 1 to 2 hr. The cells are then washed with PBS-TB and once with PBS before image acquisition. 

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