Microinjection protocol

B. Metabolic Labeling of Endogenous RNAs for Microinjection Nuclear and Cytoplasmic Microinjection Protocols... 

Da) molecules and results in analyte redistribution of less than 100 ixm. Matrix microinjection preserves tissue integrity, overcomes laser spot size imposed resolution limits, and has analyte redistribution that is limited to the size of the microinjected matrix droplet ( 10 xm). The sensor controlled aerosol and matrix microinjection protocols could conceivably be combined with matrix solution fixation. 

Cell microinjection is a very delicate technique, requiring considerable expertise, that can hardly be conveyed by a mere description of the technique itself. Since this is not within the scope of this book, the interested reader is thus referred to more comprehensive reviews, describing in details various equipment and protocols (Graessman et al., 1980 Wadsworth, 1999). Here, we will only give a general description of the application relative to the study of gap junctional communication between cells. 

The ability of 2,5-HD-modified tubulin generated in vitro to alter microtubule function in an in vivo model system was verified using sea urchin zygotes. Microinjection of 2,5-HD-treated tubulin into normal sea urchin zygotes before the first mitotic cycle caused obvious abnormalities, including small spindles, abnormal chromosomal movement at anaphase, and poor cytokinesis. Depending on the protocol used, mitosis was either grossly disrupted or simply slowed (42). 

Due to their large size, amphibian oocytes can be easily microinjected. This can be exploited to label the NPC labeled in vivo according to the following general protocol (for further details, see Pante et al., 1994) ... 

The protocol described below allows characterization of the tip diameter by bubble pressure measurement (Mittman et al., 1987). This is a nondestructive method for determining tip size, and micropipettes can still be used for microinjection experiments without any restrictions. This is not the case when micropipettes are characterized by electron microscopy. 

The speed and efficiency of the automated injection system allow biochemical analysis of microinjected cells (Pepperkok et al, 1993b). Between 500 and 1000 cells that have been labelled with [ S]methionine or are sufficient to analyze labeled proteins by gel electrophoresis. The following protocol is routinely used in our lab for this purpose. It may have to be modified according to the specific requirements of the biological experiments. 

As with other in vitro systems, different mRNAs exhibit differing translational efficiencies in the Xenopus extract. Where comparisons have been possible, relative translational yields in the extract reflect those seen on microinjection into Xenopus oocytes. Natural mRNAs normally translate best, while the performance of most synthetic mRNAs is much enhanced when transcribed from the vector pSP64T (Krieg and Melton, 1984), where the cDNA is flanked by sequences from the Xenopus P-globin cDNA. A reliable protocol for the synthesis of capped transcripts in vitro is given in Matthews and Colman (1991). 

BARS concentration by some 5-fold to 15-fold, based on the calculations that the intracellnlar concentration is around 20 //g/ml and on the assumption that 5-10% of the cell volume is injected. Prior to injection, the protein is mixed with 0.4 mg/ml fluorescein isothiocyanate (FITC)- or tetramethyl-rhodamine B isothiocyanate (TRITC)-labeled dextran (Molecnlar Probes) as a marker for the microinjected cells (Bonazzi et al., 2005). To give an example, in stndies of basolateral and apical transport (using the vesicular stomatitis virus glycoprotein and p75, respectively), the proteins were microinjected 1 h after the beginning of the 20° incubation in the transport assay. After injection, the cells were allowed to recover for 1 h before proceeding with the experimental protocol (Bonazzi et al, 2005). The BARS (p50-2) and dynamin (DYN2) antibodies were injected at 4.5 mg/ ml, 3 h before farther experimental procedures. 

This chapter discusses protocols for fluorescent labelling of antibodies, optimized fixation, and permeabilization of cells, and describes several immunostain-ing procedures with the aim to quantitatively anafyse the localization of antigens in fixed and living cells. Various aspects of the imaging equipment presently available, and the ways how to most efficiently use it in order to quantify and document results will be discussed. Labelling in living cells by microinjection of fluorescently labelled antibodies and subsequent time-lapse microscopy will also be discussed. 

The methods presented herein are histology compatible protocols for use with either proteins (Methods 1 and 3) or smaller molecules, including peptides, lipids, and drugs and their metabolites (Methods 2 and 3). These protocols include matrix solution fixation (Section 3.1) a sensor controlled aerosol (Section 3.2), and matrix microinjection (Section 3.3), a novel method for labeling single cells. 

Quality control methods for each protocol are also presented. Applications and representative data are presented for the sensor controlled aerosol and matrix microinjection, whereas representative data for matrix solution fixation are reported elsewhere (8). Sensor controlled aerosol is applied to multimodal cell tracking by fluorescence and multiple reaction monitoring (MS/MS) of a fluorescent dye incorporated into stem cells and tumor cells surgically implanted into a mouse brain. This protocol enables validation of MSI using confocal microscopy for the tracking of implanted cells, and potentially the effect of a given cell s microenvironment upon its molecular composition. Matrix microinjection is used to acquire a MALDI mass spectrum of a single motor neuron from layer V of a mouse motor cortex. 

A superior strategy to the above approach is provided by Cui and Paul [18]. The quality of this work is enhanced by the full description of the polymers that were utilized and the complete characterization of the composites that were prepared. The PP that was employed in this study was Pro-Fax PH020 manufactured by Basell. MAPP (PP-g-MA) was PB3200 provided by Cromption with a MA content of 1.0 wt.%. The diamine and montmorillonite (Cloisite Na) were identical to the ones utilized in the above study. Cloisite 20A was utilized as a control organomontmorillonite. This is a superior choice to the organomontmorillonite employed in the studies above. The composites were prepared with a DSM Micro 5 compounder. The test samples were prepared with a DSM microinjection molder. This is a superior protocol in relation to the compression-molded test samples prepared above. PP-g-MA was reacted directly with the diamine in a Brabender at 195°C and 50 r/min for 5.5 min. The amine... 

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