Immunolocalization using either fluorescence for light microscopy (LM) or gold particles for electron microscopy (EM) has become a common tool to pinpoint proteins involved in recombination during meiotic prophase. Each method has its advantages and disadvantages. For example, LM immunofluorescence is comparatively easier and higher throughput compared to immunogold EM localization. In addition, immunofluorescence has the advantages that a faint signal can often be enhanced by longer exposure times and colocalization using two (or more) probes with different absorbance and emission spectra is straightforward. However, immunofluorescence is not useful if the object of interest does not label with an antibody probe and is below the resolution of the LM. In comparison, immunogold EM localization is higher resolution than immunofluorescent LM localization, and individual nuclear structures, such as recombination nodules, can be identified by EM regardless of whether they are labeled or not. However, immunogold localization has other disadvantages including comparatively low signal-to-noise ratios, more difficult colocalization using gold particles of different sizes, and the inability to evaluate labeling efficiency before examining the sample using EM (a more expensive and time-consuming technique than LM). Here we describe a method that takes advantage of the good points of both immunofluorescent LM and EM to analyze two classes of late recombination nodules (RNs), only one of which labels with antibodies to MLH1 protein, a marker of crossovers. The method can be used readily with other antibodies to analyze early recombination nodules or other prophase I structures.
Recombination nodule Immunofluorescence Electron microscopy Chromosome Meiosis Recombination
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Supplemental Fig. 2View of two aluminum plates with plastic slide box for 25 glass microscope slides located between the aluminum electrodes for glow discharge. The aluminum electrodes (plates) are insulated from each other by a Lucite rod, and the lower plate has short plastic legs to isolate the apparatus from the floor of the vacuum chamber. Each aluminum plate is connected by a wire to the vacuum-tight electrical plug in the lid of the vacuum chamber. It doesn’t matter which plate is the cathode and which is the anode, but they need to be electrically isolated from each other (PDF 110 kb) (JPEG 110 kb)
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