DOI: 10.6075/j09s1r4f
title: CIL:39162, mouse, medium spiny neuron. In Cell Image Library

description: Imaging mode: Single tilt
description: Experiment #5DAT KO mouse 04/22/03

Description: Photoconverted dye-filled striatal medium spiny 
neurons for EM

Animal Info:ID#wt3wt4
 Weight: 34g32g
 DOB: 9/30/029/30/02
Protocol

1. Perfusion (at Duke) 
Nembutal; 4% paraformaldehyde + 0.1% gluteraldehyde

2. Sectioned on Vibratome (at NCMIR)
Thickness = 100 microns
Store in 1X PBS in fridge

3.Fill cells with Lucifer yellow
4.Store slices with filled cells in 4% para in fridge

5.Wash 6x with PBS 1X (on ice)

6. When ready to begin photoconversion, turn on the chiller in 
confocal room. Set at ~4?C. The refrigerator unit should be set 
at TEMP &lt; 45?C. Switch ON. Stage needs around 20 minutes to 
come to temperature. Pull unit out into hallway (to avoid 
increase in temperature).

6.Place slices in 2% glut/PBS on ice for 15 minutes

0.8 ml 25% gluteraldehyde
2 ml 5x PBS
6.2ml ddH20

7.Briefly wash slices in PBS
8.Place slices in PBS/glycine for a few minutes

38 mg glycine
10 ml 1x PBS

9.Follow instructions for Photoconversion of Lucifer Yellow-
filled cells
10. After photoconversion, remove DAB solution and wash slice 3x 
10 minutes in generous volumes of PBS on ice. Must remove all 
DAB before beginning osmification.

Microwaving protocol for osmication, dehydration, and embedding 
of photoconverted slices

*Prepare Resin mix and let it sit covered and undisturbed 
until needed (instructions by fume hood in embedding area).

*Rinse slices with a generous amount of cold 1X PBS on ice 
for ~ 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 following 
dilutions:

90% ethanol
70% ethanol
50% ethanol

*Make up osmium solution under fume hood and chill on ice

*1% osmium tetroxide in PBS on ice.

2.0 ml PBS 5X
then 5.5 2x distilled H2O
2.5 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 40 
seconds 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)


1st
2nd
50% EtOH


70% EtOH


90% EtOH


100% EtOH


100% Acetone


*All of the dehydration steps should be carried out in 
microcentrifuge 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 15 
min
1:1 Resin:acetone

* Check rear water load at 
7.5 minutes

Switch to 100% resin for 3 x 10 minutes:

1st
2nd
3rd
100% Resin

*Periodically check rear water 
load

*Flat embed samples between mould release slides and place 
in embedding oven under vacuum.
description: Maximum intensity project of a tomographic reconstruction of a spiny dendrite from a 4 ?m thick section throuh medium spiny neuron of mouse caudateputamen