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1.
It is currently a routine practice to require a measurement of a housekeeping reference, including actin, glyceraldehyde-3-phosphate dehydrogenase, β-tubulin, among others, in Western blots, as it is the rule in RNA blots. Reversible Ponceau staining has been applied successfully to check equal loading of gels. Here we test a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and housekeeping protein immunodetection under different conditions. Our results show that Stain-Free gels outperform Ponceau staining and that both are more consistent than housekeeping proteins as a loading control.  相似文献   
2.
We have examined the expression of actinidin, a cysteine protease found in kiwifruit, over the course of fruit development. Protease activity was first seen in fruit that had reached about half their final weight, and rose to high levels at harvest. The 5-flanking region (nucleotides –1301 to +58) of a kiwifruit actinidin gene was fused to the -glucuronidase (GUS)-coding region, and the chimaeric gene was introduced into transgenic petunia plants. Induction of the GUS gene was observed during the later stages of seed pod development, closely resembling the pattern of actinidin induction in fruit tissues of kiwifruit. Some GUS expression was also detected in the vascular system of the receptacle, leaves, stems and roots. A shorter promoter fragment consisting of nucleotides –115 to +58 conferred similar spatial and temporal regulation in some of the transgenic plants.  相似文献   
3.
The Hoechst dye staining method has been successfully applied to the central nervous system in mammals and its use has been demonstrated in intracerebral transplantation. The technique is rapid, simple and based on intrinsic nuclear properties. It was found to be permanent and valid whatever the animal strains or ages, allowing the distinction of rat cells from those of mouse, studied either separately or in a cross-transplantation model. It permitted the detection of grafted cells in the area of transplantation and the observation of early dispersion around the implantation site. Moreover, it can be combined with immunohistochemistry as demonstrated by a myelin marker in a relevant model. Immunodetection can thus help to directly observe grafted cells, at distance from the locus of transplantation, confirming their presence in the graft-type myelin patches.

Because of its rapid performance, this technique can be used systematically after transplantation to check for the presence of grafted cells in the host.  相似文献   
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The structure of the vacuolar ATPase from mesophyll tonoplasts of Mesembryanthemum crystallinum has been studied by electron microscopy using negatively stained specimens of membrane-bound and detergent-solubilized ATPase molecules. We observed a high density of particles on the surface of tonoplast vesicles and “head and stalk” structures on the edge of the membrane, similar to the F0F1-ATPases of mitochondrial and chloroplast membranes. The staining conditions, which are often critical for such small objects, were improved by using methylamine tungstate as negative stain for the membrane-bound ATPase. Compared to other staining solutions generally applied, dissociation of the F1-like enzyme complex from the membrane was best prevented and structural damage of the vesicles was least observed with methylamine tungstate. In freeze-fracture electron microscopy of tonoplast vesicles, where dissociation never occurs since no detergent is used, we also observed “head and stalk” structures on the edge of the membranes, beside many particles on the fracture faces. The detergent-solubilized ATPase forms string-like structures, caused by the aggregation of the hydrophobic membrane-embedded F0-like part of the enzyme. After negative staining the F1-like enzyme complex is arranged alternately along both sides of the string and connected by a narrow stalk.  相似文献   
6.
采用TUNEL染色及免疫组织化学技术对光化学法脑缺血后细胞凋亡及其相关基因bcl-2表达的变化进行了研究。结果发现,缺血后12h,损伤侧皮层缺血区内凋亡细胞数及bcl-2免疫反应阳性细胞数明显增加,一直持续至缺血后72h;并呈现下列时程变化:在缺血后3h每张切片上几乎无凋亡细胞出现,以后逐渐增加,缺血后12h达到峰值,缺血后24h和缺血后72h逐渐减少,但仍高于假手术组水平。凋亡相关基因bcl-2的表达在缺血后3h以前不明显,缺血后12h逐渐增加,缺血后24h最多,以后逐渐下降。上述结果提示,缺血后凋亡细胞的时程变化可能与缺血后梗塞灶的发生和发展有关,而bcl-2表达的变化可能与抑制细胞凋亡、发挥内源性细胞保护作用有关。  相似文献   
7.
The wheat grain is the most important organ for human food and therefore is the target for much research focused on modifying its composition to improve nutritional and functional components. Genetic transformation provides a precise tool to alter the composition of wheat grain by expressing new genes or by down-regulating groups of proteins encoded by multigene families such as gliadins, which contain clusters of epitopes that are active groups in triggering celiac disease. For such work, specific promoters are required to express such constructs in the wheat endosperm. In the present study we report the isolation and characterization of a γ-gliadin promoter from transgenic wheat, and the analysis of gliadin synthesis during grain development in bread wheat by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI/TOF MS). The γ-gliadin promoter fragment was isolated from bread wheat by genome walking and was re-introduced, driving the expression of the gusA gene, by particle bombardment, giving fifteen independent transgenic lines. Detailed analysis of the sequence of the 885 bp promoter fragment showed that it contains three prolamin boxes but only one is conserved according to the consensus sequence reported. The AACA/TA motif is present twice in published γ-gliadin promoter sequences. The RY element i.e., CATGCAT or CATGCAC, is also present twice in the published promoter. Transgenic lines were classified as high, medium, and low expressers. The expression of the gusA gene was found only in the seeds of the transgenic lines. GUS staining was first detected in the outer endosperm of the lobes, and then it extended to the whole outer endosperm. GUS staining was not found in the aleurone layer nor in the embryo. The qRT-PCR data confirmed the data obtained by GUS staining. The expression of the gusA gene determined by qRT-PCR for the high expresser line (B281) was 4 and 8 times higher than that of medium (B282) and low (B286) expresser lines, respectively. MALDI/TOF-MS showed that gliadins exhibited different patterns of synthesis during the course of seed maturation. Thus, gliadins with masses higher than 36,000 Da were synthesised within the first 12 days post anthesis while those with masses lower than 36,000 Da were synthesised later. Results of GUS staining, qRT-PCR and MALDI/TOF-MS showed that the γ-gliadin promoter reported in this work could be a good candidate to downregulate wheat gliadins.  相似文献   
8.
In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells [45, 46]. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5 regions were translationally fused with the -D-glucuronidase reporter gene (GUS). The GNT1 5 region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5 regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.Department of Plant Molecular Biology, Leiden University  相似文献   
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10.
In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non–small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the interactions of multiple expressed proteins within a single region of interest, and enable quantitative assessment of biomarkers in cancer cells.  相似文献   
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