Immunofluorescence staining

Cytokinesis (cell division) in animal cells involves the progressive formation in telophase of a furrow between the two daughter cells in the equator of the mitotic spindle. Immunofluorescent staining of the cortical cytoplasm at the site of the contraction ring reveals an abundance of actin as well as myosin, a-actinin, and filamin (Fishkind and Wang, 1995). Cytokinesis is highly sensitive to actin-myosin inhibitors such as cytochalasin and phalloidin. 

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

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Bakkus, M.H., Brakel-van Peer, K.M., Adriaansem, H.J., Wierenga-Wolf, A.F., van den Akker, T.W., Dicke-Evingep M.J., and Benner, R. (1989) Detection of oncogene expression by fluorescent in situ hybridization in combination with immunofluorescent staining of cell surface markers. Oncogene 4,1255-1262. 

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

Ladies, G.S., et al., Phase two of an interlaboratory evaluation of the quantification of rat splenic lymphocyte subtypes using immunofluorescent staining and flow cytometry, Toxicol. Methods, 8, 87, 1998. 

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. 8.5 Schematic presentation of indirect multiple immunofluorescence staining using hapteny lated primary antibodies (AB) raised in the same host species...

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 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.

Figure 7. Talin-containing streak-like focal adhesion plaques (A) and beta-actin cytoskeleton (B) in vascular smooth muscle cells in cultures derived from the rat aorta. Immunofluorescence staining, the nuclei couterstained with propidium iodide (A) or Hoechst 33342 (B). Microscope Olympus IX 50, digital camera DP 70, obj. 100, bar=20 pm.

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].

Figure 29. Fiuman osteoblast-like MG 63 cells in cultures on material surfaces modified with carbon nanoparticles. A fullerene Cgo layers deposited on carbon fibre-reinforced carbon composites (CFRC), B fullerene C o layers deposited on microscopic glass coverslips, C terpolymer of polytetrafluoroethylene, polyvinyldifluoride and polypropylene, mixed with 4% of single-wall carbon nanohorns, D the same terpolymer with high crystalline electric arc multi-wall nanotubes, E diamond layer with hierarchically organized micro- and nanostmcture deposited on a Si substrate, F nanocrystalline diamond layer on a Si substrate. Standard control cell culture substrates were represented by a PS culture dish (G) and microscopic glass coverslip (FI). Immunofluorescence staining on day 2 (A) or 3 (B-Fl) after seeding, Olympus epifluorescence microscope IX 50, digital camera DP 70, obj. 20x, bar 100 pm (A, C, D, G,H)or 200 pm (B, E, F) [16].

Fig. 2 Whole-mount T4 Immunofluorescence staining superposed on brightfield illumination of thyroid follicles in (a) 5 dpf control (0.1% DMSO used as a vehicle control), (b) 1.5 mM MMI-treated larvae, (c) 1 pM amiodarone-treated larvae, and (d) 50 nM T3-treated larvae. Heads of representative larvae are shown in ventral view with the anterior part on the left. Abbreviation e eye. Reprinted with permission from [46], Copyright 2009 American Chemical Society.

Frisoni GB, De Leo D, Rozzini R, et al Psychic correlates of sleep symptoms in the elderly. Int J Geriatr Psychiatry 7 891-898, 1992 Fritschy J, Benke D, Mertens S, et al 5 types of type A GABA receptors identified in neurons by double and triple immunofluorescence staining with subunit specific antibodies. Proc Natl Acad Sci USA 89 6726-6730, 1992 Frye CA, Duncan JE Progesterone metabolites effective at the GABAa receptor complex attenuate pain sensitivity in rats. Brain Res 643 194-203, 1994 Erye CA, Cuevas CA, Crystal S, et al Diet and estrous cycle influence pain sensitivity in rats. Pharmacol Biochem Behav 45 255-260, 1993 Erye PE, Arnold LE Persistent amphetamine-induced compulsive rituals response to pyridoxine (B6). Biol Psychiatry 16 583-587, 1981... 

Miller, B.M., Zitzelsberger, H.F., Weier, H.-U.G. Adler, I.-D. (1991) Classification of micronuclei in murine erythrocytes immunofluorescent staining using CREST antibodies compared to in situ hybridization with biotinylated gamma satellite DNA. Mutagenesis, 6, 297-302... 

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. 

Add 5 jjL of PI stock solution to each tube after immunofluorescence staining. 

Accessibility and preservation of the nuclear epitope to produce well-defined immunofluorescent staining patterns with minimal autofluorescence... 

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