Single through focus slice through a medium spiny neuron from the neostriatum of a wild type mouse injected with Lucifer Yellow and photoconverted.
Full resolution image description
Zip file containing through focus series in BioRad PIC and tiff format
Animation through the slices of a through focus series of a medium spiny neuron from the neostriatum of a wild type mouse injected with Lucifer Yellow and photoconverted. The flocculent material surrounding the cell likely represent photoconverted mitochondria or just background from the photoconversion procedure.
Manual tracing of dendrites and dendritic spines using Neurolucida. These files were produced in order to situate the correlated electron microscopic volumes into their approximate cellular locations; these are not meant to be highly accurate drawings.
Segmentation file description
Tar file containing the ascii file output by Neurolucida and a VMRL rendering of the final segmentation, along with the measurement summary files generated by Neuroexplorer for each part of the neuron.
Experiment #5 DAT KO mouse 04/22/03Description: Photoconverted dye-filled striatal medium spinyneurons for EMAnimal Info: ID# wt3 wt4Weight: 34g 32gDOB: 9/30/02 9/30/02Protocol1.Perfusion (at Duke)Nembutal; 4% paraformaldehyde + 0.1% gluteraldehyde2.Sectioned on Vibratome (at NCMIR)Thickness = 100 micronsStore in 1X PBS in fridge3.Fill cells with Lucifer yellow4.Store slices with filled cells in 4% para in fridge5.Wash 6x with PBS 1X (on ice)6.When ready to begin photoconversion, turn on the chiller inconfocal room. Set at ~4?C. The refrigerator unit should be setat TEMP < 45?C. Switch ON. Stage needs around 20 minutes tocome to temperature. Pull unit out into hallway (to avoidincrease in temperature).6.Place slices in 2% glut/PBS on ice for 15 minutes0.8 ml 25% gluteraldehyde2 ml 5x PBS6.2ml ddH207.Briefly wash slices in PBS8.Place slices in PBS/glycine for a few minutes38 mg glycine10 ml 1x PBS9.Follow instructions for Photoconversion of Lucifer Yellow-filled cells10. After photoconversion, remove DAB solution and wash slice 3x10 minutes in generous volumes of PBS on ice. Must remove allDAB before beginning osmification.Microwaving protocol for osmication, dehydration, and embeddingof photoconverted slices*Prepare Resin mix and let it sit covered and undisturbeduntil needed (instructions by fume hood in embedding area).*Rinse slices with a generous amount of cold 1X PBS on icefor ~ 10 min.*Turn on circulating bath (over 20?C, ~ RT): water bath(left hand side) will fill.*Insert temperature probe*Fill other T-beaker with water*Set temperature to 35?C*Open new bottle of 100% ethanol and prepare followingdilutions:90% ethanol70% ethanol50% ethanol*Make up osmium solution under fume hood and chill on ice*1% osmium tetroxide in PBS on ice.2.0 ml PBS 5Xthen 5.5 2x distilled H2O2.5 ml Osmium 4%*Rinse w/ 2x distilled H2O ? 3 x 5min*Warm up microwave for 2 minutes on high*Label tubes & place in rack on ice*Fill tubes with osmium solution (w/ meniscus at 0.5)*Using glass hooks, transfer slices to tubes*Remove temperature probe & set temp above 50?C.*Put rack w. tubes in for 40 sec at full power*Change rear water load in T-beaker*Change osmium solution on ice and microwave for another 40seconds at full power*Rinse samples for 2 minutes in distilled water on benchtop(at RT)*Insert petri bath with H2O under rack*Dehydration steps (2 x 40 seconds per step; all @ 35?C)1st2nd50% EtOH70% EtOH90% EtOH100% EtOH100% Acetone*All of the dehydration steps should be carried out inmicrocentrifuge tubes filled with 600 ml of solution.Temperature probe should be in petri dish and set for 35.Change water in rear water load when warm to touch.*Change from water to acetone in petri bath under rack ?check acetone bath level every 3 minutes*Infiltration steps (both @ 50?C):With a 50/50 mixture of resin and acetone:1 x 15min1:1 Resin:acetone* Check rear water load at7.5 minutesSwitch to 100% resin for 3 x 10 minutes:1st2nd3rd100% Resin*Periodically check rear waterload*Flat embed samples between mould release slides and placein embedding oven under vacuum.
Imaging product type
Iris size: 2.0; gain 75.4; offset 0.6
Diana Price, Aki Laakso, Michele Cyr, Maryann Martone, Naoko Yamada, Andrea Thor, Monica Berlanga (2003) CCDB:3379, Mus Musculus, medium spiny neuron. CIL. Dataset. https://doi.org/doi:10.7295/W9CCDB3379