Geoptimaliseerd protocol voor Retina Wholemount Voorbereiding voor Imaging en Immunohistochemistry

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Ivanova, E., Toychiev, A. H., Yee, C. W., Sagdullaev, B. T. Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry. J. Vis. Exp. (82), e51018, doi:10.3791/51018 (2013).

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Abstract

Materials

Name Company Catalog Number Comments
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

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References

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  2. 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).
  3. 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).
  4. Haverkamp, S., Wassle, H. Immunocytochemical analysis of the mouse retina. J. Comp. Neurol. 424, 1-23 (2000).
  5. 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).
  6. Pena, J. T., et al. miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat. Methods. 6, 139-141 (2009).
  7. 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).
  8. 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).
  9. 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).
  10. 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).
  11. 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).
  12. 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).

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