Standard histology protocol

For fixation of tissues, mice were deeply anesthetized with tribromoethanol (avertin) until they no longer displayed a withdrawal reflex in the hind limbs and then perfused intracardially with Bouin's fixative following a flush of the vasculature with saline solution. After soaking in Bouin's for one week to demineralize bones, tissues were dissected. Six segments of spine with axial muscles and spinal cord in situ, representing cervical, thoracic and lumbar spinal segments, were dissected. The brain was removed and sliced into 6 cross sectional pieces at the levels of olfactory lobes, frontal cortex, striatum, thalamus, midbrain, rostral and caudal medulla with cerebellum. Midsagittal slices of hind leg through the knees were prepared. Slices of basal skull through the pituitary and inner ears were taken. Both eyes, salivary glands and submandibular lymph node, trachea plus thyroid and sometimes parathyroid were removed and cassetted. A longitudinal slice of skin from the back was removed. The thymus, slices of lung, and a longitudinal slice of heart were cassetted. Similarly slices of liver through gall bladder, kidney with adrenal attached, pancreas and spleen were prepared. The stomach was sliced longitudinally to include both squamous and glandular portions. Loops of small intestine from 3 levels and slices of large intestine and cecum were removed, as were slices of urinary bladder. The whole uterus, with ovaries attached, was taken. In males testes were sliced longitudinally. The accessory male organs including seminal vesicles, coagulating gland and prostate were removed en block.

Altogether in most cases all tissue fit into a total of 10 cassettes. The cassettes were processed in an automatic tissue processor to dehydrate tissues which were then embedded in paraffin. Six micron sections were cut and stained with hematoxylin and eosin (H&E). Sections of brain and spinal cord in vertebral bones also were stained with luxol fast blue (LFB) for myelin and cresylecht violet (CV) for cellular detail.