이미징 및 면역 조직 화학에 대한 망막 Wholemount 준비를 위해 최적화 된 프로토콜
Summary
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.
Abstract
면역 조직 화학적 평가를 수행 할 때 섬세한 조직으로 작업하는 것은 복잡한 요소가 될 수 있습니다. 여기서, 우리는 달리 조직의 손상을 야기 다양한 프로토콜의 사용을 허용 면역 처리 중에 거실 고정 조직 모두에 구조적지지를 제공하는 링 – 지원 수화 PTFE 멤브레인을 이용하는 방법을 제시한다. 첫째, 이것은 망막 조직 생활에 형광 마커의 알약로드와 함께 보여줍니다. 멤브레인 지원을 주입하는 동안 조직의 무결성을 유지하고 추가 영상 또는 처리를위한 준비를 쉽게 전송을 허용하는 동안이 방법은, 대상 구조를 신속하게 시각화 할 수 있습니다.
둘째, 카르 보디이 미드로 고정 조직에서의 항체 염색을위한 절차를 설명한다. 파라 포름 알데히드 고정이 일반적이지만, 카르 보디이 미드 고정 시냅스 단백질의 시각화를 위해 우수한 기판을 제공. 카르 보디이 미드의 제한으로 인해 발생하는 고정 된 조직이 상대적으로 약해이라고하고 있지만, 이것은지지 막의 사용하여 극복된다. 망막 조직은 이들 기술을 설명하기 위해 사용되지만, 그들은 어떤 깨지기 쉬운 조직에 적용될 수있다.
Introduction
Performing immunohistochemistry in delicate intact tissues runs the risk of damage during handling and transfer. This can occur either in brain slices or other thin tissue, such as degenerated retina. Additionally, there are certain methods of tissue fixation that can be advantageous for immunostaining of neuronal structures, but result in compromised structural stability, precluding their use. A particular example of this is carbodiimide-based fixation, which is superior for staining receptors and hormones1-7 but is commonly avoided due to the instability of the fixed tissue.
Here, we describe a procedure which utilizes a hydrophilized PTFE membrane to structurally support delicate tissue, either fixed or unfixed, for a variety of staining techniques. The supporting membrane allows for the manipulation of delicate tissue before and after fixation, allowing for several steps of processing while minimizing the risk of tissue damage. Overall, this simple method to preserve tissue integrity provides the opportunity to use techniques that might otherwise be avoided. As such, this approach could also be successfully used for preparation of wide variety of tissues such as brain slices that become fragile following slicing procedures.
Protocol
Representative Results
Discussion
The hydrophilized PTFE membrane's high biocompatibility and transparency in solutions is advantageous when working with living tissue. This preparation has been successfully used in earlier work for patch-clamp recordings of light responses8-10. Here, we show how this approach can be adopted for structural analysis of the wholemount retina.
The bolus loading technique can be effectively used for labeling living tissue. This technique is aided by the structural support of the mem…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH grant R01-EY020535 (B.T.S), International Retinal Research Foundation and Karl Kirchgessner Foundation (B.T.S).
Materials
| Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE (Biopore), 0.4 μm |
Millipore | PICM01250 | |
| Insulin syringe, 1 ml | Beckton Dickinson | 309659 | |
| Scissors | Fine Science Tools | 15003-08 | dissection |
| Forceps, Dumont #55, inox | Fine Science Tools | 11255-20 | dissection |
| Cryostat | Leica | various | |
| Confocal Microscope System | Nikon | various | |
| Capillary glass | World Precision Instruments | 1B150F-4 | |
| P-97 Flaming/Brown Micropipette Puller | Sutter Instrument Co. | ||
| Picospritzer III | Parker Hannifin | ||
| Glass Bottom Culture Dishes | MatTek Corporation | P35G-0-14-C | |
| Petri dish | Falcon | 1008 | |
| Disposable Graduated Transfer Pipettes | VWR | 16001-180 | |
| Multiwell plates, 24-well | Beckton Dickinson | 351147 | |
| Cover glass, #1 | Electron Microscopy Sciences | 72200-30 | |
| Polysine adhesion slides | Electron Microscopy Sciences | 63412-01 | |
| Microscope slides | Globe | 1301 | |
| Liquid Blocker | Electron Microscopy Sciences | 71312 | |
| VECTASHIELD mounting medium | Vector Laboratories | H-1000 | |
| OCT medium | Sakura | 4583 | |
| Parafilm laboratory film | Fisher | 13-374-10 | |
| Dow Corning high vacuum grease | Sigma-Aldrich | Z273554 | |
| Mouse anti-PSD95 | Millipore | MABN68 | antibody |
| Donkey anti-mouse Alexa 568 | Invitrogen | A10037 | antibody |
| Isolectin Alexa 488 | Invitrogen | I21411 | |
| Chemiblocker | Chemicon | 2170S | |
| Triton X-100 | Sigma-Aldrich | T9284 | |
| Sulforhodamine | Sigma-Aldrich | 341738 | |
| Carbodiimide | Thermo Scientific | 22980 | EDC |
| Paraformaldehyde | Sigma-Aldrich | P6148 |
References
- Csaba, G., Kovacs, P., Pallinger, E. EDAC fixation increases the demonstrability of biogenic amines in the unicellular Tetrahymena: a flow cytometric and confocal microscopic comparative analysis. Cell Biol. Int. 30, 345-348 (2006).
- Fletcher, E. L., Hack, I., Brandstatter, J. H., Wassle, H. Synaptic localization of NMDA receptor subunits in the rat retina. J. Comp. Neurol. 420, 98-112 (2000).
- Gastinger, M. J., O’Brien, J. J., Larsen, N. B., Marshak, D. W. Histamine immunoreactive axons in the macaque retina. Invest. Ophthalmol. Vis. Sci. 40, 487-495 (1999).
- Haverkamp, S., Wassle, H. Immunocytochemical analysis of the mouse retina. J. Comp. Neurol. 424, 1-23 (2000).
- Koulen, P., Fletcher, E. L., Craven, S. E., Bredt, D. S., Wassle, H. Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina. J. Neurosci. 18, 10136-10149 (1998).
- Pena, J. T., et al. miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat. Methods. 6, 139-141 (2009).
- Tymianski, M., et al. A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments. Cell Calcium. 21, 175-183 (1997).
- Sagdullaev, B. T., Eggers, E. D., Purgert, R., Lukasiewicz, P. D. Nonlinear interactions between excitatory and inhibitory retinal synapses control visual output. J. Neurosci. 31, 15102-15112 (2011).
- Toychiev, A. H., Sagdullaev, B., Yee, C. W., Ivanova, E., Sagdullaev, B. T. A time and cost efficient approach to functional and structural assessment of living neuronal tissue. J. Neurosci. Methods. 214, 105-112 (2013).
- Yee, C. W., Toychiev, A. H., Sagdullaev, B. T. Network deficiency exacerbates impairment in a mouse model of retinal degeneration. Front Syst. Neurosci. 6, 8 (2012).
- Ivanova, E., Muller, U., Wassle, H. Characterization of the glycinergic input to bipolar cells of the mouse retina. Eur. J. Neurosci. 23, 350-364 (2006).
- Dmitrieva, N. A., Strang, C. E., Keyser, K. T. Expression of alpha 7 nicotinic acetylcholine receptors by bipolar, amacrine, and ganglion cells of the rabbit retina. J. Histochem. Cytochem. 55, 461-476 (2007).
