In situ metabolic labeling

In situ metabolic labeling to track the phosphorylation of IMPDH1 in intact retinas in dark/light conditions. Ana Méndez April 3^rd 2017

Eight six-week-old C57BL6 mice were dark-adapted overnight. 

Retinas were dissected in Locke´s medium under dim red light using a night vision dissecting scope, and placed in individual wells of  two 12-well dishes –one retina per well, using eight wells of each dish- in 600ml of Locke's buffer [10mM Hepes, 20mM NaHCO₃, 112.5mM NaCl, 3.6mM KCl, 2.4mM MgCl₂, 1.2mM CaCl₂, 0.1mM EDTA, 10mM glucose, sodium succinate, sodium glutamate, vitamin and amino acid supplement, pH7.4] containing 1mCi/ml ³²P-orthophosphoric acid [9.000 Ci/mmol, 15,5 mCi/ml NEX053010 Perkin Elmer, Waltham, MA, USA].
 

In more detail:  the Locke´s medium was added to the 12-well dishes, and the 12 well dishes were pre-equilibrated at the 5% CO₂ incubator, while the mice were sacrificed and retinas dissected under a dim red light setting in a room adjacent to the radioactivity lab.  The radioactivity lab was transformed into a dark room, with dim red lights.  Retinas were dissected in Locke´s buffer [dissection of 8 retinas, took about 20m]. Retinas were dissected by cutting arround the ora serrata and removing the cornea, and then carefully peeling the choroid/RPE away from the retina, maintaining the form of the intact retina and leaving the optic nerve in place when possible.  Retinas were transferred from one recipient to another, when needed, by grabbing the optic nerve with forceps.  The “dark condition” 12-well dish was prepared and dark samples were processed first.  For that, the pre-equilibrated dish was taken out of the incubator, and one retina was placed per individual well (8 wells).  At this point the radiactive ³²PPi (orthophosphate) [15,5 mCi/ml NEX053010MC Perkin Elmer] was added to each well.  After taking into account the activity of ³²PPi on the day of use, a volume was added per well that resulted in a final concentration of 1mCi/ml.  The 12-well dish was then placed in the 5% CO2 incubator for 90m [one hour and a half, exactly], to allow incorporation of the ³²P radionuclide into the ATP pool of retinal cells.  After the 90m at the incubator, the 12-well dish was taken out and placed over the bench at room temperature in the dark (behind protecting screens) for 5m exactly.  Retinas were immediately transferred to a 12-well dish with “cold” Locke´s buffer in the wells, and moved from well to well, three times for washing.  At these point, two retinas were pooled per Eppendorf tube containing 400ul of homogenization buffer, and homogenized using a manual homogenizer and white pestles.

Homogenization buffer: [20mM Hepes, 115mM KCl, 10mMNaCl, 10mM MgCl2, 50mM NaF, 5mM b-glycerophosphate, 1mM PMSF, 1 table Complete Mini, pH7.2].  At this point, samples were kept in ice, during the processing of the “light-condition” sample.
 

The whole procedure was repeated with another 8 retinas, in an identical manner until the incubation of the 12-well dish in the 5% CO2 incubator for 90m exactly.  After the 90m at the incubator, the 12-well dish was taken out and placed under over the bench at room temperature under light exposure (2000 lux white light) for 5m exactly.  Retinas were immediately transferred to a 12-well dish with “cold” Locke´s buffer in the wells, and moved from well to well, three times for washing.  At these point, two retinas were pooled per Eppendorf tube containing 400ul of homogenization buffer, and homogenized using a manual homogenizer and white pestles.
 

Samples were then centrifuged at 14.000 rpm for 20m, 4^oC, to obtain supernatant and pellet fractions. Pellets were resuspended in HB with 1% TritonX100.  Immunoprecipitation of IMPDH1 was carried out with 10mg of anti-IMPDH1 pAb and 60ml of Dynabeads-protein G (Thermo Scientific, Waltham, MA, USA). 
 

In more detail: Samples were centrifuged at 14000rpm, 20m, 4^oC in the radioactivity lab (dim red light setting). In the meantime, a new set of labeled tubes was prepared. The cleared supernatants were transferred to this new set of tubes.  Both the pellet samples and the supernatant samples were stored overnight at -20^oC.

The following day, pellets were solubilized in 200ul Homogenization Buffer + 1% Triton X100, by adding the buffer and placing the tubes in a rotating wheel for 3h.
 

At this point, there were 4 replica “dark” samples (pellet and supernatant); and 4 replica “light” samples (pellet and supernatant).  Three dark and three light replicas, pellet and supernatant (12 tubes) were used for IMPDH1 immunoprecipitation (n=3 biological replicas); while one replica (4 tubes) for control IgG immunoprecipitation.
 

IMPDH1 immunoprecipitation:  720ul of magnetic bed slurry were pre-equilibrated with homogenization buffer, 3 times.  The pre-equilibrated 720ul of magnetic bead slurry were mixed with 140ug of anti-IMPDH1 Ab, and the mix was kept at the rotating wheel for 20m at room temperature. The beads were then washed 2x with homogenization buffer.  The mix was resuspended in 1,2 ml of homogenization buffer. 100ul of this mix were added per sample tube.
 

IgG control immunoprecipitation: 240ul magnetic bead slurry was pre-equilibrated with homogenization buffer, 3x.  240ul of pre-equilibrated magnetic bead slurry were mixed with 40ug of anti-IgG Ab, and the mix was kept at the rotating wheel for 20m at room temperature. Beads were then washed 2x with homogenization buffer, and resuspended in 400ul.  100ul of this mix were added per control sample tube.
 

Immunoprecipitation protocol:
 

100ul of anti-IMPDH1-beads or anti-IgG-beads were added per sample. Volume was brought to 800ul by adding either homogenization buffer (supernatant samples) or homogenization buffer with 1% Triton X100 (pellet samples), and the binding step was allowed to proceed at the rotating wheel for 1h at room temperature.
 

The magnet was used for capture of the magnetic beads.  Unbound material was transferred to clean tubes and kept, until the final analysisas done.  Beads were washed 3x.  Elution was performed with 20ul of SDS loading buffer (Laemmli buffer).
 

Samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes, that were directly exposed to an X-ray film (AGFA Healthcare NV, Mortsel, Belgium) and subsequently immunoblotted for IMPDH1 [anti-IMPDH1 pAb used as primary Ab; goat-anti Rabbit IREDye 800 (LI-COR, Lincoln, NE, USA) as secondary Ab; and bands visualized using an Odyssey Scan System (LI-COR)].  An immunoprecipitation control was carried with an anti-IgG isotype control.