저자는 그녀의 실험실 전문 수지 퍼가기와 MS Stephania 무덤에 그녀 전문 숄 수 코넬의 도움을 인정하고 싶습니다. 저자는 자신의 조언을 교수 프랭크 Kandziora 및 박사 마리 앤 Polboth 감사하고 싶습니다.

<ol><li> 조직은 10 % 인산을 포함하는 밀봉된 컨테이너가 포르말린 용액 3 버퍼에 저장됩니다. fluorochrome 분석 fluorochromes가 빛을 구분으로 수행할 수있다면, 빛을 최소한의 노출을 보장합니다. </li><li> 필요하게 될 조직의 어느 방향 수립. </li><li> 크기로 시편을, 트림 올바른 방향으로, 밴드를 사용하여 보았다. 안전 보장 조치을 준수하고 있습니다. 표본은 다음 불투명 용기에 위치의 볼륨 충분한 고정을 달성하기 위해 약 10 배 정도 표본의 크기가되어야합니다. 표본은 포르말린 용액의 크기에 따라 한 사이에 두 주간의 총 유지됩니다. 이것은 테스트 및 최적화 목적으로 컨트롤 조직을 사용하여 제안합니다. </li><li> 조직이 포함 금형에 맞는 선택하고 70 % 에탄올을 포함하고있는 밀폐된 불투명 용기에 조직을 놓으십시오. </li><li> 빛과 지속적인 동요에 따라 차폐 에탄올의 농도를 오름차순으로 조직을 처리합니다. <ol class="lalpha"><li> 70% 에탄올 일주 </li><li> 80% 에탄올 일주 </li><li> 90% 에탄올 일주 </li><li> 백퍼센트 에탄올 일주 </li></ol></li><li> 조직은 다음에 다시 빛으로부터 지속적인 동요에 따라 차폐, 1 주일 butanol에서 지워집니다. </li><li> 수지와 뼈 밀도가 밀접 수지 밀도가 (8 단계 참조) 변경할 수 있습니다. 일치하는 우리는 정확한 밀도를 보장하기 위해 먼저 테스트 표본을 포함하는 것이 좋습니다해야합니다. 우리는 가벼운 현미경과 fluorochrome 분석을위한 Technovit 7100 (Heraeus Kulzer)를 사용합니다. immunohistochemistry가 필요한 경우 대체 수지는 그러한 Technovit 9100 (Heraeus Kulzer)로 사용해야합니다. </li><li> Technovit 7100 준비 솔루션이 필요 경우, 키트와 함께 제공되는 베이직 솔루션의 작은 aliquots와 혼합 수 있지만 더 이상 5 이상의 %를 준비 솔루션의 볼륨, 우리의 경험에 부드럽게 솔루션은 거의 필요하지 않습니다. 절대 에탄올과 기본 액체 Technovit 7100 동등한 부분은 빛의 노출을 최소화 24 시간 동안 미리 침투 솔루션으로와 교반과 혼합하여 조직에 적용됩니다. </li><li> 침투 솔루션은 다음 100 ML베이스 액체에 녹아있다 1g의 경화제 I을 (= 1 봉지)를 사용하여 구성되어 있습니다. 이것은 미리 침투 솔루션을 대체하고 지속적인 교반에 따라 1 주일 침투 수 있습니다. </li><li> 아래로 절단 표면 얼굴, 금형에 조직을 놓으십시오. 수지에 대한 조직 주위에 여백이 (이상 최소한 5mm)가 있는지 확인합니다, 이것은 블록의 강도를 보장합니다. 경화제 II 1 ML과 Technovit 7100 준비 솔루션의 15mls을 혼합하여 몰드에 시료 주위에 기름을 붓고 적어도 5 millimietres하여 커버. 경화 2 시간 이내에 시작되지만 밤새 완료됩니다. </li><li> 장착은 다음 Technovit 3040 (Heraeus Kulzer)을 사용하여 백업 블록을 도운하여 수행됩니다. 가루 한 부분 액체에 점성 액체를 얻는 2 부분의 볼륨 비율 Technovit 3040를 섞습니다. 블록의 기지 위에서 최소한 2 mm의 수준으로 블록의 뒷면에있는 쉬는 시간에 Technovit 3040 하거라. 약 10 분 후 금형이 떨어져 벗겨와 블록이 준비하실 수 있습니다. </li><li> 밀도 따라서 메타 크릴 레이트 수지의 절단 속성은 방에있는 주위 습도에 취약합니다. 블록 따라서 dessicator에 보관해야합니다. </li><li> 그라운드 sectioning는 두께 20-50 micrometres 사이의 큰 부분을 생산하고 있습니다. 두꺼운 섹션 밝은 형광을 생산로 (아래 부분을 참조) fluorochrome 분석에 유용합니다. macrotome에 다이아몬드 블레이드를 고정합니다. macrotome에있는 저수지 이러한 석유 정신으로 윤활유를 추가합니다. 클램프의 블록을 안전하고 블레이드에 동쪽을 향하게하다. 적절한 윤활을 보장 (섹션 약 20~30분) 천천히 발생하는 연삭 수 있습니다. 첫 번째 섹션은 절단 얼굴을 노출하고 섹션이 - 그것이 폐기되어야 orientates. </li><li> 다음 섹션은 다음 Superfrost 플러스 슬라이드에 배치됩니다. 섹션은 우리가 위에 샌드위치 가방의 일부를 삽입하여 다른 슬라이드 장소에 고정되어와 함께 그것이 평면 유지하는 것이 좋습니다 때문에 침묵하는 경향이 있습니다. 그 다음 1 시간 동안 섭씨 60-80도 사이의 인큐베이터로 배치됩니다. 이것은 메타 크릴 레이트를 부드럽게하고 섹션 평면 방식으로 슬라이드를 준수하는 데 도움이됩니다. </li><li> 부드러운 방식으로 미세 그릿 샌드페이퍼를 사용하여 필요에 따라 지상 부분은 세척 및 연마 수 있습니다. </li><li> 슬레지 마이크로톰는 얇은 섹션을 삭감하는 빛의 microsocopy위한 최상의 섹션을 제공합니다. 그것이 하드 블록을 절삭에 필요한 강성을 추가했습니다 같은 슬레지 마이크로톰을 사용합니다. 또는 전원 로타리 마이크로톰 사용할 수 있습니다. 날카로운 스테인레스 스틸 칼날을 사용합니다. 그것은 하나가 사용하는 다른처럼 날카롭게 수 있듯이 두 가지 블레이드를 가질 것이 바람직합니다. 블레이드는 블록과 45도 각도를해야합니다. 스rface의 연화가 필요한 경우 블록에 젖은 종이를 적용하여 얻을 수 있습니다. 그것은 품질 섹션을 얻기 위해 원하는 절삭 조건을 달성하기 위해 여러 섹션을 걸릴 수 있습니다. 섹션 다음 물을 욕조에 떠올 랐 이상 16으로 슬라이드에 배치됩니다. </li><li> 얼룩의 시약은 다음과 재료에 나열되어 있습니다. 프로토콜은 밴크로프트 외 4를 기반으로합니다. 때문에 두께 변화에 그것은 테스트 슬라이드에 얼룩을 최적화하는 것이 좋습니다. 랙 얼룩은 조직이 필요할 수 있습니다 평면 슬라이드에 직접 얼룩을 추가 있도록 슬라이드를 플로트가 발생할 수 있습니다. 동일한 프로토콜 또는 랙 얼룩은 조직 형태 측정에 필요한 지속적인 얼룩의 결과를 위해 필요합니다. <ol class="lalpha"><li> Haematoxylin과 Eosin <ol class="lroman"><li> Haematoxyilin 5~10분와 스테인 </li><li> 스콧의 수돗물로 잘 씻으십시오 </li><li> Eosin 오분와 스테인 </li><li> 수돗물에 세척 </li><li> 크실렌에 지우기 </li><li> 마운트 </li><li> 검색 결과 <ol><li> 뼈 같은 = 핑크 </li><li> Calcified 뼈 = 보라색 갈색 </li><li> 핵 = 파란색 </li></ol></li></ol></li><li> 본 K​​ossa <ol class="lroman"><li> 실버 질산 용액에 플레이스와 mineralized 뼈가 검은 전환까지 강한 빛에 노출 (약 10 분) </li><li> 증류수로 세 번 씻어 </li><li> 5 분 나트륨 thiosulfate와 위협 </li><li> 증류수로 세척 </li><li> Safrinin O와 Counterstain </li><li> 크실렌에 지우기 </li><li> 마운트 </li><li> 검색 결과 <ol><li> Mineralised 뼈를 = 검은색 </li><li> 뼈 같은 = 레드 / 핑크 </li></ol></li></ol></li><li> 메이슨 골드너 S Trichrome <ol class="lroman"><li> (80 % 에탄올의 90mls 20 분 25 % 암모니아 10mls) 알칼리성 알코올로 세척 </li><li> 물에 린스 </li><li> 증류수로 씻어 </li><li> 10 분 Weigert의 Haematoxylin와 스테인 </li><li> 증류수로 씻어 </li><li> 개양귀비빛의 - Fuchsin 최종 솔루션 오분와 스테인 </li><li> 1퍼센트 초산과 함께 15 초 린스 </li><li> phosphomolybdic 산성 - 오렌지 G 솔루션 5 분 스테인 </li><li> 1퍼센트 초산과 함께 15 초 린스 </li><li> 밝은 녹색 오분와 스테인 </li><li> 1퍼센트 초산 3 변경 린스 </li><li> 증류수로 씻어 </li><li> 마운트 </li><li> 검색 결과 <ol><li> Mineralised 뼈를 = 녹색 </li><li> 뼈 같은 = 레드 / 오렌지 </li><li> 핵 = 블루 그레이 </li><li> 연골 = 보라색 </li></ol></li></ol></li></ol></li><li> Fluorochrome 준비 : </li><li> 이들은 최소 2 주 간격, 천천히 정맥 주사에​​ 의해 관리됩니다. </li><li> 복용 <ol class="lalpha"><li> Calcein 그린 10 MG / kg IV </li><li> Oxytetracycline 50 MG / kg IV </li><li> 알리 자린 Complexone 30 MG / kg IV </li></ol></li><li> Calcein 그린의 준비 <ol class="lalpha"><li> 녹아까지 Calcein 그린 중 하나 그램은 1 M NaOH와 titrated 있습니다. </li><li> 산도는 산도 = 7.1-7.2까지 1% NaOH로 조정됩니다. </li><li> 필터와 빛으로부터 격리 보관 </li></ol></li><li> 알리 자린 Complexone의 준비 <ol class="lalpha"><li> 녹아까지 알리 자린 complexone 세 그램은 1 M NaOH와 titrated 있습니다. </li><li> 산도는 다음 산도 = 7.1-7.2까지 1퍼센트 HCL 또는 NaOH로 조정됩니다. </li><li> 필터와 빛으로부터 격리 보관 </li></ol></li><li> Oxytetracycline의 준비 <ol class="lalpha"><li> Oxytetracycline은 선반에서 안티 - 미생물의 약물로 사용할 수 있습니다. 아무 준비도 필요하지 않습니다. </li></ol></li></ol>

<ol>
	<li>Kerr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of Functional Histology. , 164-168 (2000).</li><li>Parfitt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+histomorphometry:+standardization+of+nomenclature,+symbols,+and+units.+Report+of+the+ASBMR+Histomorphometry+Nomenclature+Committee.">Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee.</a> J Bone Miner Res. 2, 595-610 (1987).</li><li>An, Y. H., Martin, K. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of histology methods for bone and cartilage. , (2003).</li><li>Bancroft, J. D., Gamble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and Practice of Histological Techniques. , 352-360 (2008).</li></ol>

용매 : <ul><li> 일반 10 % 포르말린, 에탄올, 크실렌 및 Butanol 산성을 버퍼 모든 조직학 등급, 유황 무료입니다. Grale 과학 PTY 주식 회사 http://www.grale.com.au </li></ul> 마울즈 : <ul><li> 멀리 껍질, 플라스틱 금형 Grale 과학 </li></ul> 슬라이드 : <ul><li> SUPERFROST 플러스 현미경 슬라이드 롬 과학 PTY 주식 회사 <a href="http://www.lomb.com.au/">http://www.lomb.com.au</a> </li></ul> 수지 : <ul><li> GlycolMethacrylate의 Technovit 7100 Heraeus Kulzer <a href="http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm">http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm</a> </li><li> 블럭 수지 Technovit 3040 조직을 블록을 백업 Heraeus Kulzer </li></ul> 얼룩 (Grale 과학에서 시약) 골드너의 Trichrome는 얼룩 </li> 1. Weigert의 철 hemotoxylin (DeGroat) <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> A) Hemotoxylin 솔루션 </td></tr><tr><td> Hemotoxylin </td><td> 1g </td></tr><tr><td> 절대 알콜 </td><td> 100mL </td></tr><tr><td colspan="2" style="font-weight:bold;"> B) 이런 솔루션 </td></tr><tr><td> 30% 수성 산화철 염화물 </td><td> 4mL </td></tr><tr><td> 염산 (집중) </td><td> 1mL </td></tr><tr><td> 증류수 </td><td> 95mL </td></tr></table> 필터링과 같은 hemotoxylin의 볼륨을 바로 전에 얼룩은 사용에 추가됩니다. 2. 개양귀비빛의 - fuchsin - azophloxin 주식 솔루션 <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> A) 개양귀비빛의 드 xylidine 솔루션 </td></tr><tr><td> 개양귀비빛의 데 xylidine </td><td> 0.75g </td></tr><tr><td> 산성 fuchsion </td><td> 0.25g </td></tr><tr><td> 아세트산 </td><td> 1mL </td></tr><tr><td colspan="2"> 믹스, 그리고 추가 증류수 (100mL) </td></tr><tr><td colspan="2" style="font-weight:bold;"> B) azophloxin 솔루션 </td></tr><tr><td> Azophloxin </td><td> 0.5g </td></tr><tr><td> 초산 </td><td> 0.6mL </td></tr><tr><td colspan="2"> 믹스, 그리고 추가 증류수 (100mL) </td></tr><tr><td colspan="2" style="font-weight:bold;"> C) 최종 얼룩 솔루션을 작동 </td></tr><tr><td> 개양귀비빛의 - fuchsin 솔루션 </td><td> 5 - 10mL </td></tr><tr><td> Azophloxin </td><td> 2mL </td></tr><tr><td> 0.2 % 초산 용액 </td><td> 88mL </td></tr></table> 3. 라이트 그린 솔루션 <table style="margin-left:40px;" border="0"><tr><td> 연두빛 </td><td> 1g </td></tr><tr><td> 초산 </td><td> 1mL </td></tr><tr><td colspan="2"> 믹스, 그리고 증류수를 (500mL) 추가 </td></tr></table> 4. Phophomolybdic 산성 - 오렌지 G 솔루션 <table style="margin-left:40px;" border="0"><tr><td> Phosphomolybdic 수 소산 </td><td> 3g </td></tr><tr><td> 오렌지 G </td><td> 2g </td></tr><tr><td colspan="2"> 증류수 500mL에 용해의와 크리스탈 티몰를 추가합니다. </td></tr></table> 본 K​​ossa는 스테인 <table style="margin-left:40px;" border="0"><tr><td> 1퍼센트 수성 실버 질산 </td></tr><tr><td> 2.5 %의 나트륨 thiosulfate </td></tr><tr><td> 1퍼센트 safranin O </td></tr></table> Alcian 파란색 얼룩 <table style="margin-left:40px;" border="0"><tr><td> Alcian 블루 8GX </td><td> 1g </td></tr><tr><td> 3퍼센트 아세트산 용액 </td><td> 100mL </td></tr></table> Hemotoxylin과 Eosin는 얼룩 <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> A) 해리스의 hemotoxylin </td></tr><tr><td> Hemotoxylin </td><td> 2.5G </td></tr><tr><td> 절대 알코올 </td><td> 25mL </td></tr><tr><td> 칼륨 알룸 </td><td> 50g </td></tr><tr><td> 증류수 </td><td> 500mL </td></tr><tr><td> mercuric 산화물 </td><td> 1.25g OR </td></tr><tr><td> 나트륨 iodate </td><td> 0.5g </td></tr><tr><td> 빙초산 </td><td> 20mL </td></tr><tr><td colspan="2" style="font-weight:bold;"> B) Eosin 솔루션 </td></tr><tr><td colspan="2"> 상업 생산 Eosin가 사용되었습니다. </td></tr></table> 모든 얼룩 염색의 분말은 귀르 염료 분말입니다. 모든 시약은 분석 시약, 그리고하지 랩 시약 Fluorochromes <table border="1" cellspacing="0" cellpadding="0"><tr><td> 제조 업체 </td><td> Fluorochrome </td><td> 카탈로그 번호 </td></tr><tr><td> 시그마 - 알드리치, 시드니 </td><td> Calcein 그린 </td><td> C0875 </td></tr><tr><td> 시그마 - 알드리치, 시드니 </td><td> 알리 자린 Complexone </td><td> A3882 </td></tr><tr><td> 인터 베트 오스트 레일 리아, 빅토리아 </td><td> Oxytetracycline (선반 항균 약물 할인) </td><td> Tradename; Engemycin 100 </td></tr></table>

undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis

Undecalcified 뼈 조직학은 뼈의 마이크로 아키텍처를 보여줍니다. 그것은 뼈 회전율이나 뼈 형성과 재흡수에 대한 중요한 정보를 제공 뼈의 mineralized과 세포 구성 요소를 모두 보여줍니다. 이것은 임상 및 연구 애플 리케이션의 다양한 엄청난 중요성을했다. 그것은 아름다운 이미지 1을 산출 등 fluorochrome 평가 및 조직 형태 측정이 같은 기술을 허용합니다. Fluorochrome 분석 칼슘에 바인딩 형광 염료가 그 주어진 시간에 mineralization의 금액의 부량을 허용 특정 시점에서 주입하는 기술입니다. 조직 형태 측정은 현미경 수준에서 뼈 부량의 과정이다. undecalcified 뼈 조직학을 수행하는 것은 기술적으로 특히 대형 표본과 도전이다. 그것은 조직학 임베디드 표준 파라핀에 사용되는 이들의 기술 변화가 필요합니다. 이 동영상은 좋은 품질의 섹션을 제작하는 과정을 설명하고이를 극복하기 위해있는 기술적인 문제와 방법을 보여줍니다. 표본 준비, 고정 및 처리는 종종 몇 주간 복용 밀도와 낮은 뼈의 투자율 상당히 긴 고정 및 처리 시간이 필요하기 때문에 그러나 다른 부드러운 조직과 유사한 방식으로, 함께 달성하고 있습니다. 퍼가기 그러한 메타 크릴 레이트 기반 수지로 뼛속까지 비슷하거나 동일한 경도와 밀도 지원 매체를 사용하여 달성하지만, 파라핀 침투와 포함 달리, 이것은 돌이킬 단계입니다. Sectioning 그러한 fluorochrome 분석과 같은 연구를위한 최적있는 두꺼운 섹션을 생산하는 연삭에 의해 얻을 수 있습니다. 이것은 가장 macrotome에 다이아몬드 블레이드를 사용하여 이루어진다. 또한, 얇은 부분은 빛을 현미경을 위해 생산 수 있으며 이것은 매우 날카로운 칼날과 슬레지 마이크로톰를 사용하여 이루어진다. 썰매 마이크로톰 큰 하드 블록에 필요한 추가적인 강도와 안정성을 제공합니다. 수지 포함된 섹션은 시연 아르 얼룩 다양한 물들일 수 있습니다.

Watch this Scientific Journal Video about Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis at JoVE.com

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified 뼈 조직학은 임상 및 연구 애플 리케이션의 다양한 중요 정보를 제공합니다. 그것은 기술적으로 특히 대형 표본과 도전이다. 이 동영상은 좋은 품질의 섹션을 제작하는 과정을 설명하고이를 극복하기 위해있는 기술적인 문제와 방법을 보여줍니다.

조직학, 조직 형태 측정 및 분석을위한 Fluorochrome Undecalcified 본 준비

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

undecalcified-bone-preparation-for-histology-histomorphometry

Research

JoVE Journal

Biology

27.1K 조회수.  Monash University. Undecalcified 뼈 조직학은 임상 및 연구 애플 리케이션의 다양한 중요 정보를 제공합니다. 그것은 기술적으로 특히 대형 표본과 도전이다. 이 동영상은 좋은 품질의 섹션을 제작하는 과정을 설명하고이를 극복하기 위해있는 기술적인 문제와 방법을 보여줍니다.

비디오: Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

연구

생물학

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

성인의 준비 Drosophila</em씬 Sectioning 및 현미경 분석을위한> 눈

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; 현장에서</em전체 마운트 태아 및 세포 조직학에서&gt; 하이브리드는 해양 Elasmobranchs을위한 적응

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

뼈 조정 배지 : 준비 및 생물 검정

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

대량 세포 계측법 분석을위한 샘플 준비

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

TEM 분석을위한 낮은 셀 번호와 샘플 준비를위한 다른 방법

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

눈 Microvessels의 질량 분석 기반 Proteomics 분석을 위한 시료 준비

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

싱크로트론 마이크로 CT를 위한 고처리량 피질 뼈 샘플 조달 및 분석을 위한 단면, 코링 및 이미지 처리 가이드

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

인간 골관절염 무릎 관절에서 조직 수집 및 RNA 추출

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...