Perfusion Conditions and Standard Tissue Processing

Perfusion Conditions

All procedures follow NIH Guidelines for the humane care and use of laboratory animals.

The brain was fixed in situ via intravascular perfusion with mixed aldehydes containing 2.5% glutaraldehyde, 2% paraformaldehyde, 1 mM CaCl2 and 2 mM MgCl2 at pH 7.4, 37 °C and 4 psi pressure under deep pentobarbital anesthesia. The brain was left undisturbed in the cranium for 1 hr, at which time the hippocampus was removed.

The tissue was then processed for serial EM using standard procedures.

Standard Tissue Processing for Serial Electron Microscopy
The tissue was rinsed 5 times in 100 mM cacodylate buffer with repeated agitation and then manually trimmed under a dissecting microscope to a region containing only area CA1. Area CA1 was soaked in 1% osmium and 1.5% potassium ferrocyanide in 100 mM cacodylate buffer, cooled in an ice bath to <15°C, and microwaved for 2.5 min at 37°C using the Pelco 3450 Laboratory Microwave Processor (Ted Pella, Inc., Redding, California). After several buffer rinses, the tissue was put into 1% osmium in 100 mM cacodylate buffer, cooled, and microwaved for 2.5 min at 37°C. Tissue was then rinsed 4-5 times in buffer and twice in water, then stained en bloc with 1% aqueous uranyl acetate while cooled on ice and microwaved for 2.5 min at 37°C, followed by 2 brief water rinses, and then dehydrated in an acetone series (50%, 70%, 90%, 100%) for 40 sec each, in the microwave oven at 37°C. Infiltration began with acetone and 1:1 Epon:Spurr's resins for 1 hr on a rotator at 25°C, followed by 2:1 acetone:Epon/Spurr's resins overnight. After replacement with fresh 100% resin for several hrs, samples were embedded in coffin molds with the dendrites orthogonal to the cutting plane. Samples were cured for 48 hrs at 60°C.

The blocks were trimmed to contain a region spanning the width of the slices and in the middle of s. radiatum midway between area CA3 and the subiculum. Then several 1µm thick, and 60nm thin, test sections were taken spanning the full width of the slices. Thick sections were stained with 1% toluidine blue to guide subsequent trimming. Thin sections were mounted on Pioloform-coated (SPI Supplies, Westchester, PA) slot grids (Synaptek, Ted Pella, Inc., Redding, CA) and counter stained with saturated ethanolic uranyl acetate, followed by Reynolds lead citrate, each for 5 min.

Sections were examined with a JEOL 1200EX electron microscope (JEOL, Peabody, MA) to choose an area midway between the air and net surfaces of the hippocampal slice for subsequent serial thin sectioning. At an optimal depth between 100 and 200µm from the cut surfaces, excellent tissue preservation was found as evidenced by well-preserved dendrites, with intact mitochondria, microtubules, and synapses, and the relative absence of dark or swollen neuronal processes.

A diamond trimming tool (EMS, Electron Microscopy Sciences, Fort Washington, PA) was used to prepare a small trapezoidal area <50µm on a side. Serial thin sections were cut on the Leica ultracut S ultramicrotome (Leica Inc, Malvern, Pennsylvania), mounted, and counter-stained as above for the test thins. Individual grids were placed in grid cassettes (Advance Microscopy Techniques, Danvers, MA), stored in numbered gelatin capsules (EMS), and mounted in a rotating stage to obtain uniform orientation of the sections on adjacent grids.

The series were photographed at 150 - 200 microns from the CA1 pyramidal cell body layer. The series of sections were photographed at 10,000x magnification. Calibration grids (Ernest Fullam, Inc., Latham, NY) were photographed with the series.