저자는 시설과 서비스 사용을 위해, 병리 및 진단 검사 의학의 UW 부에 의해 지원되는 부분 UWCCC 부여 P30의 CA014520에, 병리 실험실에서 위스콘신 중개 연구 이니셔티브의 대학 감사합니다.

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저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

단백질 발현을 평가하기위한 기존의 면역 조직 화학의 사용은 분석 22,23 주관적 반 정량 방법에 의해 제한된다. 사전 플랫폼은 바이오 마커의 발현 및 지역화의 높은 처리량 분석을 위해 만들어졌습니다. 조직과 세포 내 구획 모두의 상세한 분할은 사용자가 관심의 다른 마커와 바이오 마커의 발현, 현지화 및 공동 현지화를 연구 할 수 있습니다. 이전의 연구에서, 우리는 같은 세포 구획 18,20,21에 지역화 된 단백질을 연구하는 데 특히 IHC 및 다중 분광 영상의 유틸리티를 증명하고있다. 조직 마이크로 어레이 (24)와 결합하면, 이러한 기술은 병리학 자에 의해 수동 분석에 의해 허용 된 것보다 단백질 발현의 신속하고 객관적인 정량 허용합니다. 단백질 정량에 대한 면역 조직 화학 염색의 사용과 한 가지 문제 때문에 subjec에 결과 irreproducibility입니다분석과 기술 및 시약의 고유 한 차이점적인 성격. 단백질 발현을 측정하는 멀티 스펙트럼 영상 획득 등의 플랫폼을 사용하는 중요한 장점은 결과의 재현성이다. 12,16,25 크기 연속 형식으로 데이터를 반환 실질적 특히 대형 샘플로 작업 할 때, 작업 시간을 줄일 병리 컴퓨터 플랫폼 데이터는 병리학 수동 분석이 매우 상관. 연구는 서로 다른 멀티 스펙트럼 플랫폼 (14) 사이의 결과에서 전반적으로 높은 일치도를 보여 주었다. 더욱이, 우리는 이전에 단백질의 수가 12 조사에 변동이있는 경우에도 염색 결과는 재현성이 높은 것을 보여 주었다. 염색 및 분석 모두 자동 병리 플랫폼의 사용은 다른 에피토프으로 생성 된 항체에 따른 것과 같은 결과, 모든 불일치를 제거하지 않습니다 만,이 플랫폼은 편견과 적외선을 줄일 수 실질적 않습니다재현성은 일반적으로 면역과 관련된. 정확하고 재현성있는 결과를 반환하기위한 필수적인 프로토콜 내에서 특정 단계가 있습니다. 조직 또는 세포 내 마커가 정확한 분할 중요하다으로 단백질의 선택을 통해 적절한 실험 설계 포함한다. 양성 분석을 수행 할 때, 양성 염색에 대한 낮은 임계 값의 선택은 최종 결과에 큰 영향을 미친다. 에 대한 임계 값을 선택하는 동안 핵 전사 인자와 같은 단백질 &quot;온 / 오프&quot;더 이질 발현 된 단백질에 대한 임계 값을 어렵다 발견, 오히려 간단합니다. 이 이상적으로 보드 인증 비뇨 병리학 또는 분석을위한 최적의 임계 값을 찾을 수있는 여러 관찰자에 걸쳐 평균 점수로 공동으로 수행되어야한다. 이 기술의 현재 버전의 몇 가지 제한을 인식하는 것이 중요하다. 해제 할 때세포질 구획 미세화, 세 가지 방법을 사용할 수있다 : (1) (2) 막 특이 마커 염색하는 세포질 특이 마커 염색 핵 대 막의 세포질 등의 마커의 거리를 이용하여, (3)을 사용하여 연신 방법은 수동 핵 관련 세포질의 경계를 정의한다. 우리의 경험에서, 막 - 특정 마커를 사용하는 것은 가장 정확한 기술이다. 핵은 중앙에 위치한 경우 또는 관심있는 바이오 마커가 균일하게 세포질에 걸쳐 분산되어있는 경우 수동 그리기 방법은 일반적으로 정확합니다. 정확하게 섬유 아세포 및 평활근 세포 등 간질 세포의 세포질을 정의하는 것은 곤란 남아 실험을 설계 할 때 고려되어야한다. 이 기술의 또 다른 한계는 세포 분할을위한 핵시 의존성이다. 평면 부의 특정 세포의 핵을 제외하면,이 셀은 분석에 포함되지 않을 것이다. 이 경우 없음인접한 핵 또는 핵의 덩어리 사이 보이는 세포질은,이 종종 아니라 별개의 핵보다, 하나의 큰 핵 덩어리로 인식하고 있습니다. 일반적으로, 헤 마톡 실린의 Counterstain과는 핵 세분화와 대부분의 문제를 해결할 수 있습니다 핵 크기에 대한 최대 임계 값으로 허용 핵 분할 및 소프트웨어 설정의 조작 일반적으로 적절하다. 자동 병리 플랫폼의 최종 한계는 가난한 실험 설계에 의한 조직의 신뢰할 수없는 세그먼트입니다. 관심의 질문에 대답에 적합한 조직과 세포 바이오 마커를 선택의 중요성은 아무리 강조해도 지나치지 않을 것입니다. 우리는 우리의 대표적인 결과에서 입증 된 바와 같이 조직 분할하는 알고리즘을 만들 때, 크게 질병 상태 사이의 표현을 변경하는 상피 또는 기질 마커는 문제를 제기 할 수 있습니다. 그것은 어려운 문제는 발생 때와 같이 분석 대안이 있다는 것을 주목할 필요가있다. 예를 들어, 개별 IMAGES 수동보다는 이미지 트레이닝 세트로부터의 알고리즘을 적용하는 것보다, 모든 조직 구획의 도면에 의해 분석 될 수있다. 이것은 완벽하게 사용자가하고자하는 것과 일치한다 분할의 이점을 제공하지만, 그것은 또한 주관 도입 크게 분석을 완료하는 데 필요한 시간을 증가시킬 수있다. 적절한 실험 설계 분석을 촉진하고 조직과 세포 분할의 정확성을 극대화 할 수있는 가장 쉬운 방법입니다. 이 기술 및 프로토콜은 많은 미래의 응용 프로그램이 있습니다. 특정 질병 상태에 대한 다수의 바이오 마커는 식별하지만 검증되지 않은. 멀티 스펙트럼 플랫폼과 높은 처리량 목표 분석은 이러한 바이오 마커의 유효성 검사를 용이하게한다. 또한, 표현과 여러 단백질의 공동 지방화의 평가는 저조한 이​​해 신호 전달 경로에 대한 통찰력을 제공 할 수 있습니다. 의심 할 여지없이, 면역 staini의 분석과 관련된 고유의 주체성을 감소NG는 단백질 마커의 넓은 범위의 발현 및 위치 파악을 이해하는데 유용하다.

<html> 면역 조직 화학 (IHC)는 조직 내 단백질의 검출을위한 표준 실험실 기술이며, IHC 여전히 널리 연구 및 진단 병리학 모두에 사용된다. IHC 염색의 평가 결과의 해석에 잠재적 인 편견을 도입, 자주 반 정량적이다. 많은 반 정량적 인 접근 방식은 최종 진단 1-4로 염색 강도 및 염색 정도를 모두 통합하는 개발되었다. 다른 시스템은 더 나은 식 (5)를 국산화하기 위해 채점 강도와 세포 내 위치를 포함한다. 다수 시청자의 평균 점수의 혼입은 종종 단일 뷰어 바이어스 (6)의 효과를 최소화하기 위해 이용된다. 이러한 노력에도 불구하고, 분석 주관 여전히 7 오염의 정도를 평가하는 특히, 남아있다. 인간의 입력에서 프로토콜 표준화 및 최소화 주관 정확하고 재현성 IHC 결과를 만드는 중요합니다. 콘텐츠 &quot;&gt; 조직 내 단백질 발현을 결정하기위한 IHC 이외에 다른 옵션이있다. 이러한 면역과 같은 다른 기술은 단백질 발현을 조사하는 금 표준으로 간주되는 동안 연구 설정 내 면역 전통적 단백질 지역화 8을 조사하는 수단으로 간주되어 . 결정 조직 또는 세포 구획 별 발현 조직 슬라이드 형광 항체의 사용은 합리적인 타협을 제공한다. 이러한 세포 분획 또는 레이저 캡처 미세 절제 9,10와 같은 고급 기법을 도입하지 않고 어렵지만 의한 NADPH에 배경 형광도, lipofuscins, 망상 섬유, 콜라겐과 엘라스틴은 11 정확한 정량 어렵게 만들 수 있습니다. 자동 계산 병리 플랫폼은 병리 염색 12-15의보다 객관적인 정량을위한 유망 방향입니다. 조직 마이크로 어레이와 멀티 스펙트럼 영상을 결합큰 샘플 크기에서 단백질 발현의 높은 처리량 분석을 용이하게한다. 범주 형 포맷 (16)보다 연속적으로 데이터를 리턴하는 동안 실질적으로 고유 편향 및 분석에 필요한 시간의 양을 줄이면서 이러한 기술로 단백질 공동 지역화 염색 얼룩이, 조직 및 세포 내 편재 분석이 가능하다. 따라서, 본 연구의 목적은 멀티 스펙트럼 영상 소프트웨어를 사용하여 분석 면역 다중화를 수행하는 유틸리티의 방법을 설명 하였다. 이 프로토콜은 네 개의 최적화 된 단일 클론 항체와 단일 조직 섹션 슬라이드 설명서, 멀티 플렉스 면역 조직 화학 염색을 위해 작성되었습니다. 대표 실험으로서, 핵 항 - 토끼 에스트로겐 수용체 알파 (ERα) 및 안드로겐 수용체 (AR)은 막 - 결합 항 - 마우스 CD147 및 막 - 결합 항 - 마우스 E-cadherin과 함께 다중화된다. 선택의 항체는 SUBSTITUT 할 수있다여기에 나와있는 항체 에드하지만, 항체의 각 조합은 별도의 최적화가 필요합니다. 모든 항체에 대한 사전 처리는 동일해야합니다. 아칸소와 CD147 항체 칵테일로 개별적으로하고 최적화해야합니다. 각 항체는 비오틴없는 중합체 시스템 및 4 고유 발색제 중 하나를 사용하여 검출된다.

<table border="0" cellpadding="0" cellspacing="0" width="945">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylene</td>
			<td style="width:127px;">Fisher Chemical</td>
			<td style="width:119px;">X3F1GAL</td>
			<td style="width:483px;">NFPA rating:Health &#8211; 2, Fire &#8211; 3 , Reactivity-0</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethyl Alcohol-200 proof</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">4355223</td>
			<td>NFPA rating:Health &#8211; 0, Fire &#8211; 3 , Reactivity-0</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris Base</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-500</td>
			<td>NFPA rating:Health &#8211; 2, Fire &#8211; 0 , Reactivity-0</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris Hydroxymethyl aminomethane HCl</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">BP153-1</td>
			<td>NFPA rating:Health &#8211; 2, Fire &#8211; 0 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20</td>
			<td style="width:127px;">Chem-Impex</td>
			<td style="width:119px;">1512</td>
			<td>NFPA rating:Health &#8211; 0, Fire &#8211;1 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate-buffered saline</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">BP2944-100</td>
			<td>NFPA rating:Health &#8211; 1, Fire &#8211;0 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peroxidazed</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">PX968</td>
			<td>Avoid contact with skin and eyes. May cause skin irritation and eye damage.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Diva Decloaker&#160;</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">DV2004</td>
			<td style="width:483px;">This product has been classified as non&#8208;hazardous based on the physical&#160; and/or&#160; chemical nature and/or concentration of ingredients.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Estrogen Receptor alpha</td>
			<td style="width:127px;">Thermo Fisher Scientific-Labvision</td>
			<td style="width:119px;">RM9101</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Androgen Receptor</td>
			<td style="width:127px;">SCBT</td>
			<td style="width:119px;">sc-816</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD147</td>
			<td style="width:127px;">Biodesign</td>
			<td style="width:119px;">P87535M</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">E-cadherin</td>
			<td style="width:127px;">Dako</td>
			<td style="width:119px;">M3612</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Renoir Red Andibody Diluent</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">PD904</td>
			<td>It is specially designed to work with Tris-based antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DeCloaking Chamber&#160;</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">Model DC2002</td>
			<td>Take normal precautions for using a pressure cooker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Barrier pen-Immuno Edge&#160;</td>
			<td style="width:127px;">Vector Labs</td>
			<td style="width:119px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Denaturing Kit-Elution step</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">DNS001H</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mach 2 Goat anti-Rabbit HRP Polymer</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">RHRP520</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mach 2 Goat anti-Rabbit AP Polymer</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">RALP525</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mach 2 Goat anti-Mouse HRP Polymer</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">M3M530</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Betazoid DAB Chromogen Kit</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">BDB2004</td>
			<td style="width:483px;">1. DAB is known to be a suspected carcinogen. 
			2. Do not expose DAB components to strong light or direct sunlight. 
			3. Wear appropriate personal protective equipment and clothing. 
			4. DAB may cause sensitization of skin. Avoid contact with skin and eyes. 
			5. Observe all federal, state and local environmental regarding disposal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Warp Red Chromogen Kit</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">WR806</td>
			<td style="width:483px;">Corrosive. Acid that may cause skin irritation or eye damage.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vina Green Chromogen Kit</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">BRR807</td>
			<td>Harmful if swallowed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bajoran Purple Chromogen Kit</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">BJP807</td>
			<td>Flammable liquid. Keep away from heat, flames and sparks. Harmful by ingestion or absorption. Avoid contact with skin or eyes, and avoid inhalation.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Cat Hematoxylin</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">CATHE</td>
			<td style="width:483px;">Purple &#160;solution &#160;with &#160;a &#160;mild &#160;acetic &#160;acid &#160;(vinegar) &#160;scent. &#160;May &#160;be 
			&#160;irritating &#160;to &#160;skin &#160;or &#160;eyes. &#160;Avoid &#160;contact &#160;with &#160;skin &#160;and &#160;eyes. &#160;Avoid &#160;ingestion.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">XYL Mounting Media</td>
			<td style="width:127px;">Richard Allen</td>
			<td style="width:119px;">8312-4</td>
			<td>NFPA rating:Health &#8211; 2, Fire &#8211; 3 , Reactivity-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 Coverslips</td>
			<td style="width:127px;">Fisher Brand</td>
			<td align="right" style="width:119px;">22266858</td>
			<td>Sharp edges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubation (Humidity)Chamber</td>
			<td style="width:127px;">obsolete</td>
			<td style="width:119px;">obsolete</td>
			<td>Multiple vendors available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Convection Oven</td>
			<td style="width:127px;">Stabil- Therm</td>
			<td style="width:119px;">C-4008-Q</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Background Punisher Blocking Reagent</td>
			<td style="width:127px;">Biocare Medical</td>
			<td style="width:119px;">BP974</td>
			<td>This product is not classified as hazardous.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">inForm software</td>
			<td style="width:127px;">PerkinElmer</td>
			<td style="width:119px;">CLS135781</td>
			<td style="width:483px;">Primary multispectral imaging software used in manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuance software</td>
			<td style="width:127px;">PerkinElmer</td>
			<td style="width:119px;">NUANCEEX</td>
			<td>Software used for making spectral libraries within manuscript</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectra microscope and slide scanner</td>
			<td style="width:127px;">PerkinElmer</td>
			<td style="width:119px;">VECTRA</td>
			<td>Automated slide scanner and microscope for obtaining IM3 image cubes</td>
		</tr>
	</tbody>
</table>

quantitation of protein expression and co-localization using multiplexed immuno-histochemical staining and multispectral imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

도 1에서, 트레이닝이 아닌 조직 함과 함께, 상피 및 간질 부분으로 분할 이미지 전립선 조직에 대해 수행된다. 상피 막 마커 E-cadherin의를 사용하여 세포 분할이도 2에 도시 된 핵, 세포질 및 막 부분을 분리 하였다. 한 실험에서는도 3에 도시 한 바와 같이, AR, ERα, E 헤린 및 CD147의 발현 및 위치 파악을 조사 다중화 IHC를 사용 하였다. 이러한 기술을 사용하여, 우리는 ERα 모두 핵 발현 양성 세포를 식별 할 수있다 AR (그림 3B - 3C)도 3a에 녹색 화살표와 같이 비색 신호를 중첩에도 불구하고. 우리는 홍보의 다른 상태에서 더블 긍정적 인 기질 세포의 비율에 현저한 차이를 발견ostate 질환 (그림 3D). 우리는 마커 단백질 (그림 3A에서 빨간색 화살표)로 E-cadherin의를 사용하여 CD147의 세포막 - 특이 적 발현을 정량, 우리는 전립선 조직에서 막 특정 CD147의 발현을 조사하는 최초의 그룹이었다. 우리는 전립선 암의 진행 (도 3E)에 관련 CD147의 발현이 유의하게 감소하는 결과 및 수술의 예후 (19)의 중요한 관계를 발견 하였다. 가난한 실험 설계가 부정확 조직 세분화로 이어질 수있는 경우가 있습니다. 도 4에서, 상기 알고리즘은 조직의 집합에 적용하지 않았다 정확하게 세그먼트 상피 및 간질 구획 (도 4C). 이 실험에서, α-평활근 액틴 (α-SMA)은 간질 함을 표시하기 위해 사용되었다. α-SMA 및 평활근은 Figu (일부 종양 감소 또는 손실되기 때문에) 4A 재, 조직 세분화 (그림 4B)를 통해 생성 알고리즘은 정확하게 혼자 형태 계측 학적 데이터로부터 상피와 기질 구획을 구별 할 수 없습니다. 조직 또는 세포 구획을위한 단백질 마커를 선택할 때주의해야합니다. <img alt="그림 1" src="/files/ftp_upload/53837/53837fig1.jpg" /> 그림 1 : 멀티 스펙트럼 이미징 소프트웨어를 사용하여 조직의 분할. 전립선 조직 마이크로 어레이는 안드로겐 수용체 (AR), 에스트로겐 수용체 알파 (ERα), E-cadherin의, CD147 및 다중화를 이용하여 면역 염색 하였다. 트레이닝 이미지의 집합 조직 유형 및 이미지의 전체 세트 (A)의 질환 상태를 나타내는 멀티 스펙트럼 영상 획득 소프트웨어에 반입 하였다. 조직 카테고리는 기질 (녹색), 상피 세포 (적색), 및 비 - 조직 (청색)를 포함하여 생성하고, 카테고리는 수동 위에 드로잉에 의해 규정 된교육 이미지 (B). 교육 이미지를 그린 후, 조직 세분화하는 알고리즘 제작 한 이미지 (C)의 훈련 집합에 적용. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53837/53837fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53837/53837fig2.jpg" /> 그림 2 : 핵, 세포질 및 막 구획에 셀 분할. 핵 구획 헤 마톡 실린 대조 염색 (A)에 대한 최소 임계 값을 설정하여 셀 분할에 대해 정의되었다. 세포질은 소프트웨어 (B) 내에서 미리 설정된 알고리즘을 이용하여 핵 관련하여 정의 하였다. E-cadherin의 멤브레인 마커로 사용하고 최소 OD 임계치 막 실 (C)을 정의하는 도포 의미한다. 이 기술은 모든모든 세포 내 구획 (D)에서 단백질 발현의 동시 정량 OWS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53837/53837fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/53837/53837fig3.jpg" /> 그림 3 : 공동 현지화 및 멤브레인 관련 단백질 발현 분석. 다중화 IHC는 안드로겐 수용체 (AR), 에스트로겐 수용체 알파 (ERα), E-헤린 및 CD147 (A)의 발현 및 위치 파악을 조사 하였다. DAB (브라운 원체)은 AR (B) 및 ERα (C)를 ​​표시하는 데 사용 붉은 색 원체를 표시하는 데 사용 하였다. 우리는 ERα와 AR의 핵 공동 현지화와 세포의 비율을 (A 녹색 화살표) 공간적으로 중복되는 바이오 마커를 확인하고 (D) 정량화 할 수 있었다전립선 기질 내에서. 플라즈마 막 (A에서 빨간색 화살표)를 정의하는 E-cadherin의 (검은 색 발색)를 사용하여, 우리는 전립선 조직 (E) (19) 내에서 CD147의 막 - 특이 적 발현을 정량화. 분산 분석 (ANOVA)을 통계 분석에 사용 된 편도는, 오차 막대는 평균의 표준 오차를 반영하고, 별표가 P &lt;0.05를 나타냅니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53837/53837fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/53837/53837fig4.jpg" /> 그림 4 : 실험 설계에 의한 부적절한 조직 분할. , 안드로겐 수용체 (적색 발색), 및 안드로겐 수용체 변이 7 (DAB의 발색), 양성 및 악성 전립선 조직은 α-평활근 액틴 (녹색 발색 α-SMA)에 대한 염색 하였다. α-SMA가 사용 하였다기질 마커 및 전립선 (A)를 포함한 일부 종양에서 감소된다. 훈련을 실시 하였다 (B) 및 조직 분할 알고리즘이 적용되었을 때, 기질과 상피 구획으로 인해 α-SMA 염색 (C)의 부재에 부적절하게 분할 하였다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53837/53837fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging at JoVE.com

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

멀티 플렉스 면역 조직 화학적 염색 및 멀티 스펙트럼 영상을 사용하여 단백질 발현 및 공동 현지화의 정량

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

quantitation-protein-expression-co-localization-using-multiplexed

Nanyang Technological University

Research

JoVE Journal

Biology

12.7K 조회수.  Washington University in St. Louis School of Medicine. Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

비디오: Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

연구

생물학

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

이미징 단백질 - 단백질 상호 작용 생체내에</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

다중 Chemostat 배열의 설계 및 사용

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

대사율의 측정에<em&gt; 초파리</em&gt; Respirometry를 사용하여

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

담배 과도 식 시스템을 사용하여 MAPK 신호 전달 통로의 공간 개편 이미징

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC 프로토콜 전체 마운트 피부 준비 단일 셀 해상도에서 단백질 발현을 시각화합니다

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...