Information on our Tissue Processing Procedures
For a more detailed description of the precise methods used to process the tissue of the MBL, click here.
Fixation: Mice were anesthetized with Avertin (0.5 to 0.8 ml, i.p.) and were perfused transcardially with 0.9% phosphate buffered saline followed by approximately 15 ml of 1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M phosphate buffer. An additional 10 to 20 ml of double-strength fixative (2.5% glutaraldehyde and 2.0% paraformaldehyde in the same buffer) was subsequently injected for 1 to 2 minutes at an increased rate and pressure. The head was removed and put in the final fixative until the brain was dissected.
Tissue Processing: Tissue was embedded in celloidin (Parlodion, Fisher Scientific, P35-100, $497.43/100 g) by Dr. Glenn Rosen and colleagues at Beth Israel Deaconess Medical Center (BIDMC). Upon arrival at BIDMC, brains are placed in fresh 10% formalin for two weeks before embedding. After two weeks, the brains are washed in water for 24 hours before being dehydrated by being placed for 1 day each in a series of 80%, 95%, 100% ethanol and ethanol/ether solutions. The brains are placed into 3% celloidin (in ethanol/ether) for at least a week followed by 12% celloidin for 23 days. The celloidin block is trimmed to achieve a stable base;those brains to be cut in the coronal plane are notched over the left hemisphere, those to be cut in the horizontal plane on the left. Sections are cut on a sliding microtome at 30 µm and are segregated into 10 compartmented plastic boxes (Cat. #6636, $8.40, Brain Research Laboratories, firstname.lastname@example.org, 16179655544). These boxes have 6x6 compartments each, of which only 5x6 were used. Three brains were cut into a single boxeach brain occupying two successive rows. Each compartment therefore contained a 1-in-10 series of sections.
Sections from one compartment are then transferred as a group to one compartment of a circular net for staining. This circular net is a clear plastic cylinder divided into six (Cat. #6115, $33.75, Brain Research Laboratories) or eight (Cat. #8115, $41.20) compartments with a fine plastic mesh bottom and an open top. Every fifth section is stained for Nissl substance with cresyl violet (CV: cresylech violett, Cat No. 1A396, $222/10 g vial, Cellpoint Science, Divison of Roboz, 18009999734). This procedure entails washing sections in distilled water and then placing them in 0.5% CV solution for 35 minutes. Each section is placed in distilled water for 1 minute and then differentiated and dehydrated in 80 and 95% ethanol. A few drops of colophonium (Rosin: Sigma, R-3755) are added to the 95% ethanol baths. If differentiation is adequate the sections are then cleared with terpineol (Fisher, T31) and passed through xylol.
Every 10th section was mounted in Permount (Fisher, SP-15) on a 3x2 inch slide (Fisher, Clay Adams) and coverslipped (Fisher, Clay Adams Gold Seal 48x60 mm, 1 oz; Cat. No. 3334). Sections are mounted with careful attention to orientation so that left and right are consistently identifiable. Coronal series were mounted as if the observer were viewing the brain from the frontwith the animals left hemisphere on the right side of the slide. Horizontal sections were mounted as if the observer is viewing the brain from the dorsal surfacethe animals left hemisphere on the left side of the slide. Weights were applied to the slide for a week or more to express excess Permount and to ensure that the sections were pressed flat. Slides were carefully cleaned about three weeks after coverslipping.
The separation between adjacent sections on a single slide is 300 µm. The A and B slides are separated by four intervening sections. Usually the 1st, 11th, 21st, ... sections were mounted on Slide A, the 2nd, 12th, 22nd, ... sections were mounted on Slide C, the 6th, 16th, 26th, ... sections were mounted on Slide B, and the 7th, 17th, 27th, ... sections were mounted on Slide D. Slides A and B were sent to R. Williams, and slides C and D were retained by G. Rosen. Sections in compartments 3, 4, 5, 8, 9, and 10 were transferred to containers filled with 80% ethanol for long term storage.
Last updated July 31, 2000, Alex and Rob Williams and Glenn Rosen. Initiated April 12, 1999.