Utilizing commercial microwave for rapid and effective immunostaining.
ABSTRACT There is an accumulating literature demonstrating the application of microwaves across a wide spectrum of histological techniques. Although exposure to microwaves for short periods resulted in substantial acceleration of all procedures this technique still is not adopted widely. In part, this may be due to concerns over solutions that will avoid induction of thermal damage to the tissue when using standard microwave. Here, we offer a cooling setup that can be used with conventional microwave ovens. We utilized dry ice for effective cooling during microwave irradiation of tissue samples. To prevent overheating, the cups with tissue during exposure to microwaves were surrounded with powdered dry ice. Since the dry ice does not touch the walls of the cups, freezing is prevented. Overheating is avoided by alternating the microwave treatment with 1-2minute time periods when the cups are cooled outside of the microwave oven. This technique was used on mouse brain sections that were immunostained with microglia-specific CD68 antiserum and astrocyte labeling GFAP antibody. Both standard and microwave-assisted immonolabeling gave comparable results visualizing cells with fine processes and low background signal. Short incubation time in the microwave requires high concentrations of antibody for tissue immunostaining. We show that by prolonging the microwaving procedure we were able to reduce the antibody concentration to the levels used in standard immunostaining protocol. In summary, our technique gives a possibility to use a conventional microwave for rapid and effective immunolabeling resulting in reduced amount of antibody required for satisfactory immunostaining.
Full-textDOI: · Available from: Tibor Kristian, Mar 12, 2014
- SourceAvailable from: Tibor Kristian[Show abstract] [Hide abstract]
ABSTRACT: Mitochondria are organelles that undergo continuous cycles of fission and fusion. This dynamic nature of mitochondria is important for cell physiology. Transgenic mouse models that express mitochondria targeted fluorescence protein, in either neurons or astrocytes, were used to examine the role of alterations in mitochondrial morphology in mechanisms of ischemic brain injury. The animals were subjected to global cerebral ischemia and allowed to recover before their brains were perfusion fixed and processed for histology and confocal microscopy. After capturing z-stack images from different hippocampal sub-regions, mitochondrial organelles were 3D reconstructed using volocity software and then their morphological parameters were calculated. The data shows cell-type specific alterations in mitochondrial dynamics following ischemia. Fission is activated in all hippocampal areas at 2 h recovery with mitochondria in CA1 becoming progressively more fragmented during the 24 h recovery period. Mitochondria in CA3 and dentate gyrus neurons started to re-fuse after 24 h of recirculation; this was even more pronounced 3 days after ischemia. Astrocytic mitochondria underwent transient fission 2 h after ischemic insult and regained their normal shape at 24 h recovery. Surprisingly, no positive correlation was found between increased nitrotyrosine levels and mitochondrial fission, particularly in ischemia resistant CA3 and dentate gyrus neurons. Our data suggest that ischemia resistant neurons are able to shift their mitochondrial dynamics toward fusion after extensive fragmentation. The re-fusion ability of fragmented mitochondria is most likely a vital feature for cell survival.Journal of Bioenergetics 09/2014; 47(1-2). DOI:10.1007/s10863-014-9575-7 · 2.71 Impact Factor