이 프로젝트는 중국 국가 자연 과학 재단 (NSFC)에 의해 지원되었다 : 31301950 및 31671288입니다.

현재의 방법은 확립 수확하고 실험실에서 조직 학적 염색법 1,000 근육 샘플들을 저장하는 검증되었다. 동물의 관리 및 사용과 관련된 모든 절차는 중국의 농업의 교육부에 의해 설립 된 지침을 따랐다. 샘플 수집 1. 장비 <ol><li> 각 극저온 유리 병에 샘플 ID를 레이블. 참고 : 극저온 병은 특별히 동결 절단 (Cryosections) 절차 (그림 1A)에 대한 신선한 조직 샘플을 동결하도록 설계되었습니다. 바이알 금형 공장 (도 1C)에 폴리 프로필렌으로 제조된다. </li><li> 적절한 액체 질소 탱크에 액체 질소, 보안경 또는 안면 쉴드, 냉동기 장갑 10 개 cm 집게 25 개 cm 핀셋, 스티로폼 쿨러 (내부 치수 : 길이 20cm × 12 cm x 세로 135 cm 높이 아래의 장비를 구하는 ; 외형 치수 : 폭 X 15cm 상위 24 cm × 16 cm 길이), 플라스틱 필름 (폭 0.1 mm 두께 X 2.5 cm X 0.8 cm 길이)메스와 A4 PVC 바인딩 커버 (21cm X 29.5 cm) (도 1b). </li></ol> 근육 샘플의 제조 2 <ol><li> 앞서 시료 채취의 액체 질소로 5 분 스티로폼 쿨러를 채운다. 참고 : 액체 질소의 일부가 떨어져 종기와 신선한 샘플을 터치하면 가스로 전환하기 때문에 액체 질소의 큰 열이 필수적이다. 손실을 보충하기 위해 액체 질소의 전송의 빈도를 감소시키기 위해, 적절한 크기의 스티로폼 냉각기 샘플의 수에 따라 미리 획득해야한다. </li><li> 검체가 획득되면, 부드럽게 A4 PVC 결합 덮개의 부분에 배치하고, 근육 섬유의 방향을 관찰한다. 참고 : 여기, 우리는 돼지의 마지막 허리 뼈에 처음부터 등심의 도리스 근육의 단면을 사용했다. 시편은 약 12cm, 길이 8cm, 폭과 5cm 높다. </li><li> 시편 난을 통해 두 개의 평행 한 절개를 만들기 위해 메스를 사용하여N 근육 섬유의 방향, 길이 약 3 cm 및 0.6 cm 이격. 약 0.6 cm x 세로 0.6 cm X 1.5 cm 높이 긴 사각형으로 절개 근육 낸다. 주 : 근육 섬유의 단면적을 측정의 신뢰도는 주로 근육 절개의 섬유 배향에 의존한다. </li><li> 부드럽게 막 (도 2A)의 긴 에지에 평행 한 절개 근육의 종축과, 플라스틱 필름의 조각에 시료를 넣어 10cm 집게를 사용한다. 주 : 근육 섬유의 배향 인해 급속 동결을 시료에 균열을 방지하는 접착 기능을 고정하는 데 도움이 유지 기능 : 플라스틱 필름은 두 가지 기능을 갖는다. </li><li> 집게를 사용하여, 단지 미소 구멍 사이 표지 저온 바이알의하면 중앙으로 근육 조직을 배치하고 단단히 바이알 (도 2B)를 모자. </li></ol> 3. 냉동 및 보관 근육 샘플에스 <ol><li> 25cm 핀셋을 사용하여, 신속하게 액체 질소 (약 10-20 S)를 비등하지 않을 때까지 기다리고, 예비 냉각 스티로폼 쿨러에서 액체 질소의 전체 저온 바이알 잠수함. 참고 : 액체 질소가 끓는 때 cryovial이 너무 신중하게 10 ~ 20 초 동안 끓는 액체 질소의 병을 담가 떠. </li><li> 액체 질소 저장 탱크 또는 장기간 저장을 위해 -80 ° C에 냉동 근육 시료를 옮긴다. </li></ol> 4. 슬라이드 제조 <ol><li> -20 ℃ 및 -22 ° C의 사이에 저온 유지 장치를 사전 냉각. </li><li> 제거 이전 마이크로톰 블레이드 폐기의 저온 유지 장치에서 나이프 홀더 티롤 판을 닦아 저온 유지 장치에서 새 마이크로톰 블레이드를 설치하고, 8 μm의 절삭 두께를 설정. </li><li> 액체 질소 저장 탱크로부터 저온 바이알에서 근육 샘플을 전송 또는 -80 ° C에 냉동 저온 유지 장치로, 액체 질소 나 드라이 아이스에 반송. </리&gt; <li> 시료는 저온 유지 장치의 온도와 평형화 30 분을 기다린다. 참고 : 높은 온도가 시편에서 얼음 결정의 형성을 일으킬 수 있기 때문에 표본을 만지지 수있는 장비는 사전 냉각해야합니다. </li><li> 수용성 글리콜 및 수지 OCT ()의 최적 절단 온도 제제 하에서 시험편 홀더에 샘플을 탑재. 8 μm의 단면을 절단. 주 : CSAF 근육 섬유의 단면적이기 때문에, 근육 섬유의 방향에 대해 수직으로 절단 각도를 조정한다. </li><li> 방 온도하는 마이크로의 중심을 향해 절을 마운트합니다. 즉시 -80 ° C 냉장고에 슬라이드 상자에 슬라이드를 놓습니다. 슬라이드 RT에서 건조하는 것을 허용하지 않습니다. </li></ol>

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관심 없음 충돌은 금융 또는 다른 저자에 의해 선언되지 않습니다.

여기서는 동결 근육 기능의 조직 학적 평가를 수행하는 근육 조직을 저장하는 새로운 다공 cryovial을 설명한다. 이 프로토콜의 핵심 변성 단계는 다공 cryovial의 샘플은 직접 액체 질소에 침지된다는 점이다. 우리의 지식에, 이것은 기존의 동결 방법 중 근육의 냉동 절편 우수한 냉동 샘플 (대표 결과를 참조)를 얻을 수있는 간단하고 rapidest 방법입니다. 근육 연구자들이 직면 한...

골격 근육은보기의 영양 및 처리 지점에서 고기를 생산하는 동물의 가장 중요한 구성 요소입니다. 근육 성장의 효율성과 결과 고기의 품질 : 육류 산업에서,이 개 특히 중요한 측면이있다. 근육의 주성분으로, 근육 섬유 성장 성능과 동물 1 신선한 고기의 품질에 직접적으로 관련이 있습니다. 예를 들어, 섬유의 총 수 (TNF)와 섬유의 단면적 (CSAF)은 주로 근육 질량과 고기의 품질을 결정; 또한, 섬유 유형 구성 (FTC)가 강하게 신선한 고기의 품질이 영향을 미칩니다. 따라서, 동물의 근육 섬유 특성의 조작은 코어 수익성 농장 1의 경쟁력을 높이기 위해 매우 효과적인 방법이다. 지금까지 여러 가지 고유 및 외부 요인은 근육 섬유 characterist를 조작 확인 된ICS 1. 이 조작은 돼지 5 소 3 미오 스타틴 유전자 양 4의 Callipyge 유전자 및 RYR1 IGF2 유전자와 같은 특정 유전자와 동물의 대상 선택을 통해 달성 될 수있다. 또한, 다이어트 제어 및 특정 호르몬 치료는 근육 섬유의 특성 (6)에 중요한 역할을한다. 따라서, 유전 및 영양 요소를 결합하는 방법은 살코기의 내용과 고기의 품질을 향상시킬 수 있습니다. 근육 섬유의 구조의 해명은 여전히 ​​도전이기 때문에, 근육 섬유에 대한 연구는 육류 산업에 제한됩니다. 근섬유 특성 등 미오신 triphosphatase 아데노신 (ATP 아제) 조직 화학 분석과 같은 방법을 사용하여 식별된다. 이 방법의 냉동 (6-8 μm의) 얇은 섹션에있는 효소는 사실에 의존근육 섬유는 화학적으로 특정 제품에 반응 할 수있다. 단, 근육의 수분 함량은 위치에 관계없이 (즉, 뒤, 복부 또는 뒷다리) (7), 돼지, 토끼, 마우스 및 인간의 75 % 이상이다. 이전에 8, 9를 설명한 바와 같이, 저온부의 준비 과정 - 유물을 동결 - 근육의 높은 수분 함량은 일반적으로 발생하는 문제가 발생합니다. 대부분의 경우, 적절하게 우리의 경험에 따르면, 도살장 생산 라인에서 근육 조직을 동결하는 것은 거의 불가능하다. 여기에 제시된 프로토콜은 높은 처리량 방식으로 냉동 절편을 위해 근육 조직을 동결하기 위해 실험실에서 사용 간단하고 효율적인 방법을 설명합니다. 이 방법의 하이라이트는 액체 질소에 냉동 플래시 근육 조직 용으로 설계된 새로운 cryovial이다. 현재 워크 플로우 동시에 조직을 용이하게 할 수명확하게 표시 함과 세포질 주변 조직에 꽉 근섬유의 동격으로 우수한 근육 동결 절단 (Cryosections)에 대한 동결 처리. 액체 질소가 조직과 혼합되지 않기 때문에, 또한,이 프로토콜은 조직 분석 옵션의 다양한 배열에 적용될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="508">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Cryostat Microtome</td>
			<td style="width:111px;">&#160;Leica</td>
			<td style="width:111px;">Leica CM1950</td>
			<td style="width:148px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Microscope&#160;</td>
			<td>Nikon</td>
			<td>Nikon DS-U3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:140px;">&#160;Cryogenic Vial&#160;&#160; Plastic film</td>
			<td></td>
			<td></td>
			<td>Designed by ourself</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cm-forcep</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25cm-tweezer</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Safety glass</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Freezer gloves</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
	</tbody>
</table>

a multi-hole cryovial eliminates freezing artifacts when muscle tissues are directly immersed in liquid nitrogen

골격 근육의 생리에 대한 연구는 적절하게 명확하게 볼 세포질 구획으로 섹션을 얻기 위해 표본을 처리하는 기술적 과제에 직면 해있다. 또 다른 장애물은 주위 조직에 근섬유의 꽉 동격이다. 조직 고정 및 파라핀 매립 처리는 근섬유의 수축을 초래하기 때문에, 동결 절편을 위해 근육 조직 강화의 최적의 방법이다. 그러나, 일반적으로 발생하는 문제, 얼음 결정의 형성은, 때문에 근육의 높은 수분 함량의 동결 절편의 준비 기간 동안 발생합니다. 여기에 제시된 프로토콜은 제 적절히 액체 질소에 침지하여 그 근육 조직을 동결하는 간단하고 효율적인 방법을 설명한다. 단독으로 액체 질소를 사용의 문제점은 다음 절연체로서 작용하고 조직의 냉각을 저해 조직에 질소 가스 배리어의 형성을 유발한다는 것이다. 하는 네브라스카이 &quot;증기 담요 &#39;효과를 방지하려면w cryovial은 조직 표면 주위의 액체 흐름 속도를 증가하기 위해 설계되었다. 이것은 병의 벽 (14 개)에 유입 구멍의 총 펀칭에 의해 달성 하였다. 버블 역학에 따르면, 작은 거품 적은 가능성의 액체 흐름의 결과의 높은 속도는 가스 배리어를 형성한다. 액체 질소가 유입 관통 구멍 cryovial로 유입되면, 조직 주변의 유속 가스 배리어를 제거하기에 충분히 빠르다. 미리 냉각 펜탄을 이용하여 근육 조직을 동결하는 방법에 비해,이 프로토콜은보다 간단하고 효율적이며 처리량하게 근육을 고정하기 위해 사용될 수있다. 또한,이 방법은 실온에서 가연성이 매우 이소 펜탄에 액세스 할 수없는 기관에 최적입니다.

cryovial 그림 및 동결 절편의 준비 기간 동안 근육을 동결에 대한 일반적인 실험실 장비는 그림 1에 나타내었다. 크리오 바이알 (cryovial)는 금형 공장에서 폴리 프로필렌으로 만들어진다. 각 바이알은 입구 구멍 (14)의 총이있다 : 하나는 캡이고; 또 하단이고; 나머지 12 형태 4 개 개의 병렬 라인, 서로 90 °의 4 개 개의 구멍 각각. 이러한 흡입 구멍 때 &quot;증기 블?...

Watch this Scientific Journal Video about A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen at JoVE.com

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

이 프로토콜은 액체 질소에 직접 급락으로 근육 조직을 동결하는 절차를 설명합니다. 이 프로토콜은 또한 질소 가스의 &quot;블랭킷 효과&quot;를 방지 할 수있는 새로운 cryovial를 강조 할 때, 액체 질소 연락처 검체의 조직 표면.

근육 조직이 직접 액체 질소에 몰입 할 때 이슈 결빙 멀티 홀 Cryovial 제거

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

a-multi-hole-cryovial-eliminates-freezing-artifacts-when-muscle

Research

JoVE Journal

Biology

11.2K 조회수.  Jiangxi Agricultural University.  이 프로토콜은 액체 질소에 직접 급락으로 근육 조직을 동결하는 절차를 설명합니다. 이 프로토콜은 또한 질소 가스의 "블랭킷 효과"를 방지 할 수있는 새로운 cryovial를 강조 할 때, 액체 질소 연락처 검체의 조직 표면. 

비디오: A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

냉동 인간 ES 세포

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

연구

생물학

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

플래시 동결 E12.5 마우스 브레인 Cryosectioning

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

, 동결 해동 및 운송을위한 포장 셀

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

인간 배아 줄기 세포를 동결 융해

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

에어 리퀴드 인터페이스에서 인간의 비강 상피 세포의 배양

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of fluorescently tagged proteins using total internal fluorescence (TIRF) microscopy is used to visualize and quantify changes in cytoskeletal dynamics. TIRF microscopy and the use of fluorescent tags also allows for quantification of the changes in cytoskeletal dynamics caused by mutations in actin. Using this technique, quantification of cytoskeletal function in live cells valuably complements in vitro studies of protein function. As an example, missense mutations affecting the actin residue R256 have been identified in three human actin isoforms suggesting this amino acid plays an important role in regulatory interactions. The effects of the actin mutation R256H on cytoskeletal movements were studied using the yeast model. The protein, Aip1, which is known to assist cofilin in actin depolymerization, was tagged with green fluorescent protein (GFP) at the N-terminus and tracked in vivo using TIRF microscopy. The rate of Aip1p movement in both wild type and mutant strains was quantified. In cells expressing R256H mutant actin, Aip1p motion is restricted and the rate of movement is nearly half the speed measured in wild type cells (0.88 &#177; 0.30 &#956;m/sec in R256H cells compared to 1.60 &#177; 0.42 &#956;m/sec in wild type cells, p &#60; 0.005).

Disease-causing mutations in actin can alter cytoskeletal function. Cytoskeletal dynamics are quantified through imaging of fluorescently tagged proteins using total internal fluorescence microscopy. As an example, the cytoskeletal protein, Aip1p, has altered localization and movement in cells expressing the mutant actin isoform, R256H.

Aip1p 역학은 굴지의 R256H 돌연변이에 의해 변경 있습니까

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

조직의 우선 순위 구분과 골격근 질환 모델에 대한 동결

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

탠덤 고압 냉동 및 투과 전자 현미경을위한 식물 조직의 빠른 동결 교체

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

플 런지 프리즈 : 투과 전자 현미경에서 서스펜션 세포의 초 구조 및 면역 로컬 ​​라이 제이션 연구를위한 도구

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

형광, 섭취 곤충에 액체 이해 능력을 결정 하기 위한 자성 나노 입자

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...