Laser capture microdissection of gonads from juvenile zebrafish

Background  

Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation.     

Laser capture microdissection of gonads from juvenile zebrafish

Background

Until recently, the limit of spatial resolution of ultrasound systems has prevented characterization of structures <1 mm. Hence, the study of ovarian follicular development in rodents has been based on one-time histological examination of excised tissues; i.e., longitudinal study of day-to-day ovarian changes has not been possible in mice and rats. The objective was to establish an ultrasonographic approach to study follicular and luteal dynamics in mice and rats.

Methods

Experiment 1 was a pilot study to develop methods of immobilization (physical restraint vs. general anesthesia) and determine technical factors affecting ovarian images using ultrasound bio-microscopy in rats vs. mice. The hair coat was removed over the thoraco-lumber area using depilation cream, and a highly viscous acoustic gel was applied while the animals were maintained in sternal recumbency. In Experiment 2, changes in ovarian structures during the estrous cycle were monitored by twice daily ultrasonography in 10 mice for 2 estrous cycles.

Results

Ovarian images were not distinct in rats due to attenuation of ultrasound waves. Physical restraint, without general anesthesia, was insufficient for immobilization in mice. By placing the transducer face over the dorsal flank, the kidney was visualized initially as a point of reference. A routine of moving the transducer a few millimetres caudo-laterally from the kidney was established to quickly and consistently localize the ovaries; the total time to scan both ovaries in a mouse was about 10 minutes. By comparing vaginal cytology with non-anesthetized controls, repeated exposure to anesthesia did not affect the estrous cycle. Temporal changes in the number of follicles in 3 different size categories support the hypothesis that follicles ≥ 20 microns develop in a wave-like fashion.

Conclusion

The mouse is a suitable model for the study of ovarian dynamics using transcutaneous ultrasound bio-microscopy. Repeated general anesthesia for examination had no apparent effect on the estrous cycle, and preliminary results revealed a wave-like pattern of ovarian follicle development in mice.     

Laser capture microdissection of mammalian tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.     

Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.     

High-quality RNA from cells isolated by laser capture microdissection

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.     

Laser capture microdissection MALDI for direct analysis of archival tissue

MALDI mass spectra were obtained from cancer cells isolated by laser capture microdissection (LCM) of archived tissue. Frozen human lung tissue from adenocarcenoma and squamous cell carcenoma cases were cut into 5 to 15 microm thick sections, stained with hematoxylin and dehydrated. Cancer cells were isolated by LCM, mixed with matrix solution, and deposited on a MALDI target for mass spectrometric analysis. For comparison with LCM isolated cells, tissue sections were placed directly on the MALDI target without microdissection. Tissue sections frozen in optimal cutting temperature (OCT) solution and cut into 8 microm thick sections gave the best performance with direct MALDI analysis. Between 15 and 20 peaks were observed in the mass region between 1,000 and 4,000 Da, and roughly half of these peaks were common to either squamous cells or adenocarcenoma. Additional peaks were observed in the non-LCM mass spectra and these may result from biomolecules in the healthy tissue. When compared to fresh tissue, both LCM and non-LCM archived tissue produced fewer peaks, possibly due to degradation of the biomolecules in the archived tissue.     

Molecular analysis of complex tissues is facilitated by laser capture microdissection

Every tissue contains heterogeneous cell populations. Laser capture microdissection (LCM) facilitates cell isolation from complex tissues followed by molecular analysis. LCM entails placing a transparent film over a tissue section or a cytological sample, visualizing the cells microscopically, and selectively adhering the cells of interest to the film with a focused pulse from an infrared laser. The film with the procured cells is then removed from the original sample and placed directly into DNA, RNA, or protein-extraction buffer for processing. LCM has revolutionized molecular analysis of complex tissues because it combines the topographic precision of microscopy with the power of molecular genetics, genomics, and proteomics. However, the success of molecular analysis still depends on the experimental design and requires the understanding of each technical step involved in specimen preparation. This review attempts to rationalize and demystify the choice of various technical options in upstream tissue processing supporting global analytical strategies.     

激光显微切割技术及其在植物学研究中的应用

激光显微切割技术是目前最为成功的解决组织中细胞异质性问题的技术。介绍了激光显微切割技术的产生和影响因素,并对其在植物基因表达分析、植物生殖和胚胎发育研究、代谢物分析以及植物与微生物互作等方面的研究做简单综述。     

Laser capture microdissection for the analysis of gene expression during embryogenesis of Arabidopsis

It is during embryogenesis that the body plan of the developing plant is established. Analysis of gene expression during embryogenesis has been limited due to the technical difficulty of accessing the developing embryo. Here we demonstrate that laser capture microdissection can be applied to the analysis of embryogenesis. We show how this technique can be used in concert with DNA microarray for the large-scale analysis of gene expression in apical and basal domains of the globular-stage and heart-stage embryo, respectively, when critical events of polarity, symmetry and biochemical differentiation are established. This high resolution spatial analysis shows that up to approximately 65% of the genome is expressed in the developing embryo, and that differential expression of a number of gene classes can be detected. We discuss the validity of this approach for the functional analysis of both published and previously uncharacterized essential genes.