Lamin antibody microinjection

Functional analyses have suggested that lamins are involved in a number of biological processes, including DNA replication, chromatin organization, cellular differentiation during development, nuclear envelope reassembly following mitosis, and the structural stabilization of the nuclear membrane during interphase (for review, see Moir et al., 1995). Here we describe two methods that we have successfully applied in our laboratories and that have allowed us to analyze functional and dynamic properties of lamins in vivo by the microinjection of cultured cells with either affinity-purified lamin antibodies or with fluorescently labeled lamins. 

II. Microinjection of Lamin Antibodies into Living Cells... 

Effects produced by microinjected lamin antibodies were monitored by immu-nocytochemistry and electron microscopy. For immunocytochemistry, after different incubation times following microinjection, cells were fixed in methanol (-20°C, 10 min) and acetone (-20°C, 1 min), and then air dried. Microinjected cells were identified by incubating the cover sUps (10 min) with antiguinea pig secondary antibodies conjugated to fluorescein isothiocyanate (FITC Dianova, Hamburg, Germany). 

In our experience, lamin antibodies do not show any observable effects when microinjected into the nucleus of interphase cells. A possible reason could be the inaccessibility of lamins to the antibodies due to the presence of condensed chromatin at the nuclear periphery. An alternative explanation is that lamin polymers are accessible to the antibodies, but that the bound antibodies do not influence interactions of lamins with other nuclear components. 

Benavente, R., and Krohne, G. (1986). Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies. J. Cell Biol. 103, 1847-18.54. 

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