Laser Ablation

General Information

The LA sample chamber can accommodate either a large solid sample (up to 2 cm thick) or a glass microscope slide. The microscope slide will have to be trimmed (with a glass cutter) to approximately 1 inch by 2 inches.

Tissue Sections

Frozen Samples:

Tissue will be fixed by perfusion for 8-24 hours using 20x volume of fixative to tissue (brain will need longer fixation). Treating the tissue with a sucrose gradient allows for easier section cutting by cryoprotecting tissue (use 10% to 30% sucrose O/N). Place your tissue in a cryomold containing OCT (optimal cutting temp) media with NO BUBBLES and orient tissue (remember the initial cutting surface is the base of the mold). Freeze on a bed of dry ice within a beaker containing cold isopentane (2-methyl butane). The cryomold will float in the isopentane (do not submerge). Samples can be stored at -80°C until ready to section. (Protocol developed by the Microscopy Core at Gladstone Institute)

Fixed Samples:

Tissue will be harvested as usual and fixed in at least 10x volume of formalin in PBS. Tissue can be stored at 4°C until sectioning.

Sectioning:

Tissue can be submitted for sectioning at the Mouse Histology And Phenotyping Core Laboratory downtown. Contact Donna Emge at d-emge@northwestern.edu or 312-503- 2679 to schedule your embedding, sectioning and mounting. All sections for LA-ICP-MS analysis should be 10-20 µm thick and unstained. It is useful to have adjacent sections stained by H&E for comparison (discuss this with the MHPL staff).

Native Agarose Gels

The gel agarose percentage and gel thickness must be optimized for high resolution separation of your protein bands. For the example in Science, the optimal gel concentration was 0.8 % w/w agarose and the optimal dimensions were 8 cm x 5.5 cm x 0.3 cm. Your running buffer will also be optimized for your proteins of interest. For the separation of Atx1-TM complex and Ccc2a, the running buffer was 20 mM Tris-HCl and19.2 mM Glycine at pH 8.5. At this pH, Atx1 migrates towards the anode and Ccc2a towards the cathode. Your gel may be Coomassie stained to locate protein bands; however, all solutions should be analyzed for metal content. The identity of the proteins present can be confirmed by ESI-MS.

Gels must be bound and dried prior to LA analysis. To do this, the gel is transferred between two sheets of cellophane which are about 3-4 cm longer than then gel. The excess solution is removed carefully, paying special attention not to break the gel or leave air bubbles. The bound gel is then placed between 3 layers of pure cellulose chromatography paper (0.19 mm thickness). Approximately 2 kg of weight are placed then on top of the gel and chromatography paper, and after 1 h, the paper sheets are replaced with fresh ones. The weight has to cover the entire gel surface to obtain uniformly dried gels. At least two more paper changes every hour, before leaving the gel to dry overnight worked best. If the gel is still not completely dry after 24 h, replace the chromatography paper and continue. The dried gel is now ready for LA-ICP-MS analysis. (Protocol developed by Dr. Monica Canalizo)

**Contact Becky for More Information**