Explant Culture Medium
45% HAMS F-12 (Gibco #11765-054)
45% DME (Gibco #10313-021)
10% FCS (HyClone)
Insulin (5 mg/ml 1000X stock in H2O with HCl)
(10 ml/liter) L-glutamine (200 mM, Cellgro # 25-005-CI)
(10 ml/liter) Penn/Strep (Cellgro #30-002-CI)
(10 ml/liter) HEPES (Cellgro #25-060-CI)
100X (cold) Thymidine
60 mM thymidine 0.145 g thymidine
up to 10 ml with 1X PBS
sterile filter and store at -20°
retinal conditioned medium (Protocol C.6)
polycarbonate filters (Corning/Nucleopore #110410, 1 micron pore size)
Kodak NTB2 emulsion (#1654433)
Dektol Developer + Rapid Fixer (#807-7927)
[3H]thymidine (NEN #NET027Z ,70-80 Ci/mmol); store in 70% EtOH at -20°
Add 16.91 g Dektol Developer to 250 ml Q at 37° while stirring
and cool to 15° prior to use.
Add 44.9 g Fixer to 250 ml Q while stirring at room temperature.
Dissect retinae in prewarmed explant culture medium.
Immediately transfer to 1 ml explant culture medium containing [3H]thymidine (5mCi/ml).
Culture for 1 hour at 37° and then wash 5 times in 5 ml prewarmed explant culture medium.
Place on a polycarbonate filter floating in 1 ml medium in a 12 well dish.
Culture for the desired number of days and every 24 hours add 20 microliters of conditioned retinal supernatant.
Or, for continuous labeling change the explant culture medium each day with medium containing fresh [3H]thymidine.
After culture, wash in PBS and dissociate as describe in protocol D.6.
Following immunohistochemistry, remove as much liquid as possible and dip in emulsion prewarmed to 42° in a slide mailer in the dark.
Dry in a light tight box in an upright position for several hours and store during the exposure time at 4° in a light tight and dessicated slide box. Usually 3-4 days is sufficient for in vitro labeling experiments.
Slowly warm the slides to room temperature and immerse in Dektol developer for 2 minutes at 15°C.
DO NOT AGITATE, the emulsion will come off.
Place gently in a water bath (room temperature) for 30 seconds and fix in Kodak Fixer for 4 minutes.
Rinse several times in Q and mount as usual with gelvatol.