Naslov
Direct visualisation of immunogold-labelled vinculin within focal adhesion sites on the undersurface of fibroblastic cells

Autori
Richards, Geoff, R ; Štifanić, Mauro

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Abstracts of European Cells and Materials I Bone & Soft tissue Biomaterial interactions / Richards, Geoff, R - Davos : AO Foundation, 2001, 16-17

Skup
European Cells and Materials I Bone & Soft tissue Biomaterial interactions

Mjesto i datum
Davos, Švicarska, 22.08.1999. - 24.08.1999

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Nije recenziran

Ključne riječi
Immunogold; Vinculin; Scanning Electron Microscope; Focal adhesion

Sažetak
An understanding of cell-substratum adhesion to biomaterial surfaces is of value in promoting the attachment of connective tissue onto implant materials. In addition, adhesion of cells to implant surfaces is one of the major factors which determines the implant’s biocompatibility. The current definition of biocompatibility is that it refers to the ability of the material in question to perform with an appropriate host response in a specific situation (Williams, 1987). In certain circumstances on implant surfaces it is important to have adhesion of the soft tissues to prevent capsule formation and in other circumstances it may be desirable to have no adhesion, such as where tendons cross implants. Therefore measuring adhesion of appropriate cells and tissues to implant surfaces is very important for helping to screen new implant surface designs. Previously a semi-quantitative method of measuring the amount of cell adhesion was shown. This was based on general heavy metal staining, scanning electron microscopy (SEM) and image analysis of both the whole cell shape and the stained areas at the cell-substrate interface(Richards et al., 1996). Also Hunter et al., 1995, studied cell attachment using indirect immunofluorescent labelling of vinculin, quantifying the degree of cell attachment by determining the mean number of focal adhesions, containing vinculin, and determining the mean total area of the focal adhesions per cell. We now report an improved method for indirect immunocytochemical labelling of the integral protein vinculin within the cell focal adhesions for SEM viewing. The focal adhesions are either viewed, with the SEM, from above with the cells still on the substrate or from below, after the cells have been embedded in resin and the substrate removed. The advantages of this method over immunofluorescent labelling are: the possibility of high resolution viewing, there is no possibility of colour fading or autofluorescence problems - samples can be kept for years since gold particles do not disappear and also because of the particulate nature quantitation can be more accurate. There are two main advantages of this new method over the standard indirect immunolabelling for SEM viewing. 1. The literature (Beesley, 1993) suggests blocking (with the non-immune serum from the species that provides the 17 secondary antibody) to be performed before applying the primary antibody. We show that non-specific labelling is lower if this is performed just before the secondary antibody is applied. 2. Detergent extraction (using Triton X-100) is performed before the fixation. This is possible since it is known that Triton X-100 doesn’t extract vinculin from focal adhesions (Niederreiter et al., 1994). By this step most of the cell upper layers are removed allowing an easy visualisation of adhesion patterns in SEM with very low background labelling. The method will now be developed with image analysis techniques to allow a more accurate quantitative comparison of cell adhesion to different materials.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA