Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 134

Mold (we found a small weigh boat works well)
Cryostat specimen disc
Microscope slides (gelatin-coated glass slides or SuperFrost Plus,
Cat# 48311-703, VWR)

In vitro Transcription kit (Cat# AM1326, Ambion)
DIG RNA labeling mix (Cat# 11 277 073 910, Roche)
Fluorescein RNA labeling mix (Cat# 11 685 619 910, Roche)
RNA Purifi cation columns (Cat# 11 814 427 001, Roche)
0.5 M EDTA pH 8.0

2.3.2 In Vitro
Transcription and
Riboprobe Purifi cation

DEPC-dH 2 O
Dilute 1:1,000 DEPC in dH 2 O, incubate at 37 °C overnight
and autoclave
4 % Paraformaldehyde/PBS (pH 7.2)
Acetic anhydride solution
To 250 ml DEPC-dH 2 O add 2.3 g NaCl and 3.7 ml
triethanolamine.
Just before use, add 1.25 ml acetic anhydride, stir vigorously.
Acetic anhydride is unstable in water and quickly hydro-
lyzes; therefore, it is important to prepare immediately
prior to use.
1:1 (v:v) acetone methanol solution chilled to 4 °C
1× prehybridization buffer
Dilute 2× prehybridization buffer (Cat# P-1415, Sigma-
Aldrich) 1:1 with formamide
Hybridization buffer
50 % formamide, 5× SSC, 0.1 mg/ml yeast tRNA, 0.1 %
Tween 20, 0.05 mg/ml Heparin in DEPC-dH 2 O
Humid chamber solution
50 % formamide, 2× SSC

2.3.3 In Situ
Hybridization

RNase A (Cat# EN0531, Fermentas) (fi nal 1 μ g/ml in 2× SSC)

2.3.4 Wash and
Fluorescent Staining

2× SSC, 0.5× SSC
1 % H 2 O 2 in 2× SSC (made fresh)
10× TBS
1.5 M NaCl, 1 M Tris base, pH 7.4
1× TBS (made from 10× TBS stock)
TBS-T
0.05 % Tween 20 in TBS