Name: high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses
Uploaded: 2016-02-18T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

이 작품은 국립 보건원의 난청 및 기타 통신 장애의 신경 질환 및 뇌졸중의 국립 연구소 (NINDS) 및 국립 연구소 (NIDCD), (NIH)의 교내 프로그램에 의해 지원되었다. 우리는 NINDS EM 시설과 지원 NIDCD 고급 이미징 코어 (코드 번호 ZIC DC 000081-03)를 감사합니다.

관리 및 동물의 처리는 NIH 동물 관리 및 사용위원회의 가이드 라인에 따라했다. 12 시간 빛 : 출생 후 하루 (P) 양측 우수한 둔덕을 통해 1-1.2 % CTB를 주입 15-21 흰쥐는 12 일 유지 하였다 어두운주기를. 1. 망막 조직 고정 <ol><li> 해부 현미경, 아주 좋은 팁, ​​가위, 셀룰로오스 필터 종이, 플라스틱 피펫과 현미경 슬라이드 2 집게 다음 재료 및 도구를 조립합니다. </li><li> 2.0 ml의 할로 탄 (흡입 마취제)와 밀폐 된 챔버에 쥐를 마취. 발가락 핀치, 또는 깜박임 반사의 부족 후 후방 발의 철회의 부족 :이 방법으로 적절한 마취를 결정합니다. 그런 다음 단두대 즉시 목을 베다. pH가 7.4에서 0.1 M 인산 완충액 (PB)에 4 % 파라 포름 알데히드를 포함하는 유리 접시에 홍채 가위 쌍의 장소 눈을 제거합니다. </li><li> 해부 현미경을 사용하면, 옥수수를 제거안구의 전면을 차단하여 개. 집게와 내부 망막 표면에서 렌즈와 유리체를 제거합니다. </li><li> 망막이 아이 컵에서 분리 될 때까지 두 집게로 공막 껍질. </li><li> 면도기 200 μm의 두께 스트립, pH의 변화 고정 대상 - (100)에 즉시 망막을 잘라. </li><li> 실온에서 20 분 - 10의 pH 10.5에서 30 분 다음에 4 % 파라 포름 알데히드를 더한 0.01 %의 글루 타르 알데히드 - 20 pH가 6.0에서 0.1 M PB 4 % 파라 포름 알데히드의 망막 스트립을 수정합니다. </li><li> (4 ℃에서의 pH 7.4), 0.15 mM의 CaCl2를 가진 PB 여러 회 세척 한 후에 0.1에서 (30 %의 O / N 후, 10 %, 20 %, 30 %로 각각 60 분) M 글리세롤 망막 스트립 cryoprotect 대체를 동결하기 전에 PB. </li></ol> 2. 동결 대체 참고 :이 동결 대체 방법은 이전에 게시 된 프로토콜 (19, 20)에서 수정됩니다. 또한, 악기 (장갑을 착용) 매우 추운 것을 매우 중요하다; 영형악기를 터치하면 therwise, 조직을 부분적으로 해동 수 있습니다. 이 단계의 모든 AFS 챔버 내에서 수행되고 악기는 챔버의 가장자리 위의 이동이 허용되지 않습니다. 유사하게, AFS에 사용 된 모든 화학 물질의 적절한 냉각이 필요하다. <ol><li> - 플 런지 동결 EM의 냉동 보존기구 (CPC)에서 -184 ° C에서 액체 프로판의 망막 스트립. 좋은 브러시를 사용하여, (브러시를 사용하여 별도의 버퍼를 심지) 급락로드의 끝에 가서 금속 스텁에 부착 된 양면 테이프의 작은 조각에 샘플을 배치합니다. </li><li> 액체 프로판으로 봉 런지 약 5 초 동안 거기에 유지하고, 액체 질소로 채워진다 작은 이송 챔버를 이용하여 자동 동결 교체기구 (AFS)으로 전송 ( &quot;LN 2 크라이 이송 컨테이너를 냉각 &#39;). </li><li> AFS에 냉동 샘플 및 기기 (집게와 메스)를 배치 한 후, 그 자체에 대한 실에서 악기를 냉각veral 분 조직을 터치하거나 작은 이송 챔버에 몇 초 동안 기기의 팁을 배치하여 이들을 냉각하기 전에 (액체 질소 충전 () LN 2 크라이 이송 컨테이너를 냉각 &#39;). </li><li> AFS에서하면 벌금 메스를 사용하여 스텁에서 샘플 테이프를 제거합니다. 또한, 질소 가스의 유량 제어를 유지 TF 이러한 과정에서 오픈 (TF는 &#39;LN이 증발기 용 조정기 제어&#39;라고 기재 함). </li><li> (AFS를 설정하기 전에, IE) 평면 매립 홀더에 고정 된 조직을 배치하기 전에, 투명 플라스틱 시트 얇은 원형 절단 각 웰의 바닥 라인하도록 홀더의 하부에 배치. 완료 할 때 중합 시험편 블록 비교적 쉽게 제거 할 수있다. 참고 : 이전에, 우리는 두 번 라이 헤르 + 젤라틴 캡슐 (Petralia와 Wenthold, 1999 참조) 사용하는 평면 삽입 홀더에 다른 방법을 사용하고, &lt;SUP&gt; 20. </li><li> = 4 ° C / 시간 (11.3 시간), T2에 의해 32 시간, S1은 = 증가 온도 T1 = -90 ° C ~ -45 ° C ~ 50 시간, S2에 대한 : AFS (악기 용어를 사용)에서 다음 자동 시퀀스를 사용하여 = 5 ° C / 시간 (9 시간), (총 = 142.3 시간) 40 시간 동안 T3 = 0 ° C로 증가 온도. 이전에 Timed 시퀀스를 시작하기 (-90 ° C에서) AFS에서 샘플을 배치하는 4 시간은 2 - 걸릴 수 있습니다. </li><li> AFS에서&gt; 32 시간 동안 -90 ℃에서 메탄올 1.5 % 우라 닐 아세테이트에 얼어 붙은 섹션을 담가. 평면 포함 알루미늄 홀더에 일곱 우물의 각 조직 (일반적으로) 두 가지를 놓고 (세 AFS에 맞게). <ol><li> 우물에서 우라 닐 아세테이트 / 메탄올로 조직 + 테이프를 놓고는 테이프에서 조직을 제거 너무 어려운 경우, 직전에 침투 (예 : Lowicryl HM20)를 시작 포함 매체에, 나중에 테이프를 제거합니다. </li></ol></li><li> 이어서, 온도를 단계적으로 증가 시간 당 -45 ° C (+ 4 ℃로; 자동 순서대로). </li><li> 각 플랫 포함 홀더에서 기존의 용액을 제거하기 위해 뾰족한 플라스틱 피펫을 사용하여, 다음 예비 냉각 신선한 메탄올을 추가하는 또 다른 표준 플라스틱 피펫을 사용하여, 미리 냉각 된 메탄올의 샘플을 세 번 씻으십시오. </li><li> 1, 2 :이어서, 동일한 방법을 사용하여 예 1의 매체 (HM20은 / 메탄올 매립 낮은 온도 매립 수지 점진적 샘플 침투 2 시간 동안 순수 수지이어서 2 시간 동안 한 각 후 수지를 변경할 등) O / N을 유지합니다. </li><li> 단지 웰의 상단에 도달하는 수지의 레벨을 조정 다음날 다시 수지를 변경한다. </li><li> 샘플을 중합 (-45 ° C 자동 시퀀스에서 0 ° C로, 시간 당 + 5 ° C) 40 시간 동안 UV 빛. </li><li> 그런 다음 AFS에서 샘플을 제거합니다. 일반적으로 샘플 블록은 여전히​​ 색상에 약간의 핑크색을 보여줍니다. RT O에서 화학 물질 흄 후드에서 형광 빛에 가까운 샘플을, 배치 / N 이상 그들이 때까지 앱귀를 완전히 취소합니다. </li></ol> 3. Postembedding EM Immunogold 면역 세포 화학 참고 : 23,24,25 설명 된대로 Postembedding의 면역 세포가 수행된다. <ol><li> 1 % 톨루이딘 블루와 1 ㎛을 Ultramicrotome와 부분, 얼룩 부분을 잘라 및 단면 방향을 위해 그들을 검사; 절편의 최적의 횡단면을 달성하기 위해 조직 블록의 방향. </li><li> 을 Ultramicrotome 70 nm 두께의 얇은 부분을 잘라 Formvar - 탄소 코팅 니켈 슬롯 그리드에 그들을 수집합니다. </li><li> 워시는 TBS 버퍼 세 번 (TBS, 0.05 M 트리스 완충액, 0.7 %의 NaCl, pH를 7.6) 세척 한 다음 증류수 H 2 O에 한 번 메쉬. </li><li> 하나 NMDAR 소단위 (항 - 토끼 GluN2A 1:50 항체 : 염소 항 - CTB를 함유하는 혼합물을 20 μL의 방울 (3000 1)의 다음, 30 분 동안 TBS 5 % BSA 20 μL 떨어진다 그리드 부화 및 , GluN2B 1:30)을, 또는 항 - 토끼 GluA2 / 3 (1:30)을2 % BSA와 TBS-트리톤 (TBST, 0.01 % 트리톤 X-100, 산도 7.6) 및 0.02 M NaN이 3 O / RT에서 N. </li><li> 5 분 TBS (산도 8.2) 다음 세 가지 별도의 10 TBS 방울 (20 μL) (PH 7.6), 10, 20 분에 세척 그리드. </li><li> TBST에 18 나노 금 입자에 결합 된 10 나노 금 입자와 당나귀 방지 염소 IgG에 (1:20)을 수행 결합 당나귀 항 - 토끼 IgG를 (1:20)를 포함하는 혼합물의 방울에 2 시간 (20 μL)에 대한 그리드를 품어 2 % BSA 및 0.02 M NaN의 3 (PH 8.2). </li><li> 초순수에 다음, 5, 5, 10 분 동안 TBS의 20 μl의 방울 (PH 7.6)에 그리드를 세척 한 다음 건조. </li><li> 어두운 조건 하에서 각각 8 및 5 분 동안 증류하여 H 2 O 5 % 우라 닐 아세테이트 및 0.3 %의 납 시트르산 Counterstain과 그리드. </li><li> 트리플 - 표지 실험, 그리드 부화 O / 항 - 염소 CTB의 혼합물 20 μL 방울 (1 : 3000)과 함께 RT에서 N, 항 - 마우스 PSD-95 (1 : 100), 및 항 - 토끼 GluN2A (1 : 50). 그런 다음 2를 2 시간 동안 그리드를 품다2 % BSA 및 0.02 M NaN이 3 TBST에 18, 10, 5 nm의 금 입자에 결합 된 IgG를 혼합 (PH 8.2) 0 μL 방울. 세척 사이에 항체 배양 후으로 대조 이중 라벨과 같은 절차를 유지합니다. </li><li> 컨트롤 이차 항체 단독인가 된 수행 하였다. </li><li> EM에보기 그리드 및 디지털화 된 이미지. 이 24 필요한 경우에만 밝기 및 대비를위한 어도비 포토샵 6.0에서 최종 수치를 처리합니다. </li></ol> 4. 정량 <ol><li> 수동 8,000X 확대에 구멍이나 균열이없는 내부 얼기 층 (IPL)의 선택 영역은, 무작위 25000 배율에서 IPL의 전체 깊이를 합성 사진. </li><li> 그들은) 역행 수송 CTB 신호 (큰 금 입자)가 포함되어, b)을 나타낸다 잘 정의 막은, 갈라진 틈 및 시냅스 밀도, C)는 적어도 두개의 작은 금 부분을 포함 할 때 원뿔 바이폴라 댓구에서 RGC 수지상 식별PSD 내의 icles 또는 extrasynaptic 세포막을 따라 하나 이상의 작은 금 입자. </li><li> 정량 분석​​을 위해 상기 기준에 따라, 55 RGC 수지상 - (45)를 수집한다. </li><li> PSD 및 NIH ImageJ에 소프트웨어 개별 RGC 덴 드라이트의 extrasynaptic 세포막의 길이를 측정한다. 같은 막 10 nm의 금 입자 내의 카운트 (7)의 평균 두께에 기초하여, 막 - 관련 - 세포막 26 9 내지. </li><li> 선형 마이크​​로 미터 (금색 / μm의) 당 금 입자의 수​​가 입자 밀도를 계산한다. 각 금 입자의 중심 (시냅스 위치) PSD의 중간 또는 (extrasynaptic 위치) PSD의 가장자리 사이의 거리를 측정한다. </li><li> 소프트웨어에 의해 통계 분석을 수행합니다. 수단을 비교하는 두 꼬리 t-테스트를​​ 사용합니다. 의미 P &lt;0.05 결론 지었다. 달리 명시하지 않으면, 리포트 값으로 ± SEM (18)를 의미한다. </li></ol>

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우리는 성공적으로 정량 후 삽입 immunogold에게 EM을 네 가지 기술을 설명했다 : 1) 짧고 약한 고정, 2) 동결 대체, 3) 포스트 포함 immunogold 염색, 4) 정량. EM의 immunogold는 얇은 조직 섹션에서 특정 단백질의 검출을 할 수 있습니다. 금 입자로 표지 된 항체를 직접 EM을 사용하여 가시화 할 수있다. 막 수용체의 subsynaptic 현지화를 검출하는 강력한 반면, EM의 immunogold 기술적으로 도전, ?...

글루타메이트는 망막 1에서 주요 흥분성 신경 전달 물질이다. 바이폴라 전지 (2)로부터 글루타메이트 시냅스 입력을 수신하는 망막 신경절 세포 (망막 신경절 세포)는, 뇌 영상 정보를 전송 망막의 출력 뉴런이다. 생리 학적 연구는 망막 신경절 세포의 시냅스 여기가 NMDA 수용체 (NMDARs)와 AMPA 수용체 (AMPARs) 3,4,5에 의해 postsynaptically 매개되는 것으로 나타났다. 망막 신경절 세포의 흥분성 시냅스 후 전류 (EPSCs)가 AMPARs 및 NMDARs에 의해 매개되어 있지만 망막 신경절 세포에 3,5,6,7,8, 자연 미니어처 EPSCs (mEPSCs)는 만 AMPARs 매개 구성 요소 4,5,9을 나타낸다. 그러나, 글루타메이트 흡수를 감소시키는 것은 RGC의 수상 돌기에 NMDARs이 흥분성 시냅스의 외부에 위치 될 수 있음을 시사 자발적인 EPSCs 5 NMDAR 구성 요소를 한 것으로 밝혀졌습니다. 막 - 관련 구아닐산 키나제 등 PSD-95 (MAGUKs)이 글루타메이트 수용체 이온 채널을 포함한 클러스터 신경 전달 물질 수용체시냅스 부위에서의도 구별 subsynaptic 10,11,12,13,14 발현 패턴을 나타낸다. 최근 수십 년간, 촛점 면역 사전 매립 전자 현미경 (EM) 면역 위에 막 수용체 발현을 연구하기 위해 사용되었다. 촛점 면역 수용체 발현 넓은 패턴을 보여준다하더라도, 그 해상도가 낮은 것이 불가능 세포 내 위치를 구별하는 데 사용할 수있다. 포유 동물의 망막에 사전 내장 EM 연구는 NMDAR의 서브 유닛이 콘 바이폴라 전지 리본 시냅스 15, 16, 17에서 시냅스 요소 내에 존재 함을 나타냅니다. 이것은 생리 학적 증거 명백 대조적이다. 그러나, 반응 생성물의 확산을 미리 매립 immunoperoxidase 방법에서 공지 된 이슈이다. 따라서,이 방법은 일반적으로 통계적으로 신뢰할 수있는 데이터를 제공하지 않습니다 및 extrasynaptic 막 18,19,20,21 대 시냅스 막에 현지화 사이의 구별을 제외 할 수 있습니다. 에다른 한편으로는, 생리 및 해부학 적 데이터는 망막 신경절 세포 3,5,7,9,22에 AMPARs의 시냅스 현지화과 일치한다. 따라서, 망막 리본 시냅스에서 글루타메이트 수용체와 MAGUKs는 시냅스에뿐만 아니라 perisynaptic 또는 extrasynaptic 막 구획뿐만 아니라 현지화됩니다. 그러나, 망막 리본 시냅스에서 이들의 막 단백질의 고해상도의 정량 분석​​이 여전히 필요하다. 여기서는 콜레라 독소 소단위 B를 사용하여 라벨 (CTB)를 래트 망막 신경절 세포 상 시냅스에서 이들 단백질의 개수 밀도 변동을 추정 하였다 NMDAR 소단위, AMPAR 소단위와 PSD-95 subsynaptic 현지화를 검사 postembedding EM의 immunogold 기술 개발 역행 추적 방법.

<table><tbody><tr><td>Paraformaldehyde</td><td>EMS</td><td>15710</td><td></td></tr><tr><td>Glutaraldehyde</td><td>EMS</td><td>16019</td><td></td></tr><tr><td>NaH2PO4</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Na2HPO4</td><td>Sigma</td><td>7782-85-6</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C-8106</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A-7030</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T-8787</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>221465</td><td></td></tr><tr><td>NaN3</td><td>JT Baker</td><td>V015-05</td><td></td></tr><tr><td>Glycerol</td><td>Gibco BRL</td><td>15514-011</td><td></td></tr><tr><td>Lowicryl HM 20</td><td>Polysciences</td><td>15924-1</td><td></td></tr><tr><td>Tris-Base</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>Tris</td><td>Fisher</td><td>04997-100</td><td></td></tr><tr><td>Anti-GluN2A</td><td>Millipore</td><td>AB1555P</td><td>Dilution 1/50</td></tr><tr><td>Anti-GluN2B</td><td>Millipore</td><td>AB1557P</td><td>Dilution 1/30</td></tr><tr><td>Anti-GluA2/3</td><td>Millipore</td><td>AB1506</td><td>Dilution 1/30</td></tr><tr><td>Anti-PSD-95</td><td>Millipore</td><td>MA1&amp;ndash;046</td><td>Dilution 1/100</td></tr><tr><td>Donkey anti-rabbit IgG-10 nm gold particles</td><td>EMS</td><td>25704</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-10 nm gold particles</td><td>EMS</td><td>25814</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-5 nm gold particles</td><td>EMS</td><td>25812</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-goat IgG-18 nm gold particles</td><td>Jackson ImmunoResearch</td><td>705-215-147</td><td>Dilution 1/20</td></tr><tr><td>Formvar-Carbon coated nickel-slot grids.</td><td>EMS</td><td>FCF2010-Ni</td><td></td></tr><tr><td>Uranyl acetate</td><td>EMS</td><td>22400-1</td><td></td></tr><tr><td>Methanol</td><td>EMS</td><td>67-56-1</td><td></td></tr><tr><td>Lead citrate</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM AFS</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM CPC</td><td>Leica</td><td></td><td></td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td></td><td></td></tr><tr><td>JEOL 1200 EM</td><td>JEOL</td><td></td><td></td></tr><tr><td>liquid nitrogen&amp;nbsp;</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>Propane</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>CTB</td><td>List Biological Laboratories</td><td>104</td><td>1 - 1.2%</td></tr><tr><td>Anti-CTB</td><td>List Biological Laboratories</td><td>703</td><td>Dilution 1/4,000</td></tr></tbody></table>

high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

이전 24, 25 설명한 바와 같이 여기에 제시된 결과는 쥐의 망막에서 RGC의 수상 돌기에 현저하게 다른 subsynaptic 현지화 GluA 2/3의 패턴과 NMDARs을 보여줍니다. RGC 수지상 정보에 GluA 2/3 immunogold 입자의 77 %가 가장 중심 시냅스 마찬가지로, PSD (도 1a) 내에 위치 하였다. 그러나 NMDARs은 하나 시냅스 또는 extrasynaptically에 있었다. GluN2A의 immunogold 입자의 83 %가 PSD ?...

Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

망막 리본 시냅스에서 막 수용체의 고해상도 양이 Immunogold 분석

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

high-resolution-quantitative-immunogold-analysis-membrane-receptors

Research

JoVE Journal

Neuroscience

7.5K 조회수.  National Institutes of Health. The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

비디오: High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Quantifying 시냅스 : 시냅스 번호를 수치 Immunocytochemistry 기반 어세이

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

연구

신경과학

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF 현미경과 망막 바이폴라 세포 이미징 Exocytosis

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

생물학

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Hippocampal 슬라이스 문화의 시냅틱 단백질의 포스트 퍼가기 Immunogold 라벨링

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Wholemount 쥐의 망막에서 면역 조직 화학 및 칼슘 이미징 방법

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Optogenetic 및 동결 골절 복제 Immunolabeling는 마우스 편도체에서 글루타메이트 수용체의 입력 특정 배열을 검사하기 위해 결합

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

인간 면역학적 시냅스 이미징

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

면역학 및 감염병학

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

이미징 및 면역 조직 화학에 대한 망막 Wholemount 준비를 위해 최적화 된 프로토콜

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

<meta charset="utf8"></head><body>TEM을 위한 망막 오가노이드 시료 전처리에 최적화된 프로토콜

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

<meta charset="utf8"></head><body>저자 스포트라이트: TEM을 사용하여 성숙한 망막 오가노이드의 시냅스 초구조 분석

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

투과 전자 현미경을 위한 망막 오가노이드 시료 준비

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

망막 리본 시냅스에서 막 수용체의 고해상도 양이 Immunogold 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

Quantifying 시냅스 : 시냅스 번호를 수치 Immunocytochemistry 기반 어세이

TIRF 현미경과 망막 바이폴라 세포 이미징 Exocytosis

Hippocampal 슬라이스 문화의 시냅틱 단백질의 포스트 퍼가기 Immunogold 라벨링

Wholemount 쥐의 망막에서 면역 조직 화학 및 칼슘 이미징 방법

Optogenetic 및 동결 골절 복제 Immunolabeling는 마우스 편도체에서 글루타메이트 수용체의 입력 특정 배열을 검사하기 위해 결합

인간 면역학적 시냅스 이미징

이미징 및 면역 조직 화학에 대한 망막 Wholemount 준비를 위해 최적화 된 프로토콜

투과 전자 현미경을 위한 망막 오가노이드 시료 준비

투과 전자 현미경을 위한 망막 오가노이드 시료 준비

투과 전자 현미경을 위한 망막 오가노이드 시료 준비