Novel method for immunofluorescence staining of mammalian eggs using non-contact alternating-current electric-field mixing of microdroplets

Shirasawa, Hiromitsu; Kumagai, Jin; Sato, Emiko; Kabashima, Katsuya; Kumazawa, Yukiyo; Sato, Wataru; Miura, Hiroshi; Nakamura, Ryuta; Nanjo, Hiroshi; Minamiya, Yoshihiro; Akagami, Yoichi

doi:10.1038/srep15371

Cited by 20 publications

(34 citation statements)

References 25 publications

(48 reference statements)

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“…Both procedures remove the highly abundant lipids in myelin-rich brain tissue [ 39 ], which is an important step to obtain optically transparent tissues, but also improves antibody penetration. Improvement of antibody penetration is important because immunofluorescent staining of large tissues is still a challenge [ 40 , 41 ]. Furthermore, we noticed that certain antibodies require further optimization for staining of large tissues.…”

Section: Discussionmentioning

confidence: 99%

Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment

Lagerweij

Dusoswa

Negrean³

et al. 2017

Angiogenesis

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Background Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and migration of cells in the brain of mice, however, with limited imaging depth. To enable comprehensive analysis of GBM and the brain microenvironment, in-depth 3D imaging methods are needed. Here, we employed methods for optical tissue clearing prior to 3D microscopy to visualize the brain microvasculature and routes of invasion of GBM cells. Methods We present a workflow for ex vivo imaging of optically cleared brain tumor tissues and subsequent computational modeling. This workflow was used for quantification of the microvasculature in relation to nuclear or cellular density in healthy mouse brain tissues and in human orthotopic, infiltrative GBM8 and E98 glioblastoma models. Results Ex vivo cleared mouse brain tissues had a > 10-fold imaging depth as compared to intravital imaging of mouse brain in vivo. Imaging of optically cleared brain tissue allowed quantification of the 3D microvascular characteristics in healthy mouse brains and in tissues with diffuse, infiltrative growing GBM8 brain tumors. Detailed 3D visualization revealed the organization of tumor cells relative to the vasculature, in both gray matter and white matter regions, and patterns of multicellular GBM networks collectively invading the brain parenchyma. Conclusions Optical tissue clearing opens new avenues for combined quantitative and 3D microscopic analysis of the topographical relationship between GBM cells and their microenvironment.

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Section: Discussionmentioning

confidence: 99%

Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment

Lagerweij

Dusoswa

Negrean³

et al. 2017

Angiogenesis

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“…For example, IVM makes it possible to study the expression of the proteins that are related to the aging of oocytes, such as cohesin protein, in human oocytes of various stages. Using new technology like electric‐field mixing technology for efficient immunofluorescence staining, research on the aging of oocytes with IVM will advance the field further.…”

Section: Recent Research On Fertility Preservation Using In Vitro Matmentioning

confidence: 99%

In vitro maturation of human immature oocytes for fertility preservation and research material

Shirasawa

Terada

2017

Reprod Medicine & Biology

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AimIn recent years, the importance of fertility preservation (FP) has increased. In vitro maturation (IVM), an important technique in FP, has started to be used in the clinic, but controversies persist regarding this technique. Here, a survey of IVM for FP is provided.MethodsBased on a literature review, the applications of FP, methods of FP, IVM of oocytes that had been collected in vivo and ex vivo, maturation of oocytes after IVM for FP, cryopreservation of oocytes for FP, explanation of the procedures to patients, and recent research on FP using IVM were investigated.ResultsAlthough IVM for FP remains controversial, the application of FP is expected to expand. Depending on the age and disease status of the patient, various methods of oocyte collection and ovarian stimulation, as well as various needle types and aspiration pressures, have been reported. The maturation rate of IVM in FP ranges widely and requires optimization in the future. In regard to cryopreservation for matured oocytes, the vitrification method is currently recommended.ConclusionRegarding FP for patients with cancer, the treatment of cancer is prioritized; thus, the time and use of medicines are often constrained. As several key points regarding IVM remain unclear, well‐designed and specific counseling for patients is necessary.

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“…Data on the BMI of the 14 patients in this study are shown in Tables and . The average BMI was 27.0 ± 7.5 kg/m 2 , which is categorized as overweight . There was no significant correlation between the number of collected oocytes and BMI, as shown in Figure C ( R 2 = 0.105, P = 0.257).…”

Section: Resultsmentioning

confidence: 90%

“…After IVM, the maturation status of oocytes was assessed using immunofluorescence staining with a previous technique. 21,29 In brief, the oocytes were fixed with 3.7% formaldehyde and incubated for 15 minutes in 0.2% Triton X-100. To detect microtubules, the primary antibody was mouse monoclonal anti-β-tubulin antibody…”

Section: Immunofluorescent Staining Of Oocytesmentioning

confidence: 99%

Oocyte collection and in vitro maturation after train transportation of human follicular fluid aspirated from resected non‐stimulated ovaries of patients with endometrial adenocarcinoma

Shirasawa

Ono

Kumazawa

et al. 2019

Reprod Medicine & Biology

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Purpose Immature human oocytes from resected ovaries can be used for research and fertility preservation, though it is unknown whether it is feasible to transport oocytes for these purposes. This study examined in vitro maturation (IVM) outcomes after the transportation of human follicular fluid (HFF) containing oocytes. Methods Fourteen patients with endometrial adenocarcinoma were enrolled. Oocytes obtained from the resected ovaries of seven patients were transported with HFF by railway (transportation group). Samples of HFF from the other seven patients were not transported, and IVM was performed promptly (non‐transportation group). The results of oocyte retrieval and IVM were compared. Results The average ages in the transportation and non‐transportation groups were 40.1 ± 2.0 and 39.6 ± 1.8 years, respectively, and the average numbers of collected oocytes were 8.1 ± 8.4 and 5.1 ± 5.1, respectively. There was a significant negative correlation between the number of collected oocytes and age. The proportions of oocytes that reached meiosis II (maturation rate) after IVM were 38.6% and 69.2% in the transportation and non‐transportation groups, respectively ( P = 0.013). Conclusion In this preliminary study, the usefulness of the transportation of HFF was limited. Further studies on maintaining oocyte normality during transportation are necessary for becoming the effective method for research and clinical use.

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Novel method for immunofluorescence staining of mammalian eggs using non-contact alternating-current electric-field mixing of microdroplets

Cited by 20 publications

References 25 publications

Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment

Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment

In vitro maturation of human immature oocytes for fertility preservation and research material

Oocyte collection and in vitro maturation after train transportation of human follicular fluid aspirated from resected non‐stimulated ovaries of patients with endometrial adenocarcinoma

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