Protocol T.3

[3H]Thymidine Labeling

 

 

Solutions

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

(Sigma #T9250)

up to 10 ml with 1X PBS

sterile filter and store at -20°

Other Supplies

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°

Dektol Developer

Add 16.91 g Dektol Developer to 250 ml Q at 37° while stirring

and cool to 15° prior to use.

 

 

Fixer

Add 44.9 g Fixer to 250 ml Q while stirring at room temperature.

 

 

Procedure

• Dissect retinae in prewarmed explant culture medium.

• Immediately transfer to 1 ml explant culture medium containing [3H]thymidine (5 mCi/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.