抄録
The effects of several antigen retrieval methods on immunostaining of NOSs in paraffin-embedded rat brain tissues were studied in combination with the influence of two fixatives, 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (pH7.4), and 70% ethanol. The first antibodies for NOSs were purchased from Affinity Bioreagents Co., Ltd., New Jersey. Without any antigenretrieval treatment, paraffin sections of the rat brain fixed with both PFA and ethanol were never immunostained. Only the ethanol fixative was useful for antigen retrieval in immunostaining of NOSs. In antigen retrieval with microwave heating, retrieval solutions such as water (except the one containing 6M of urea), Tris-HCl buffers at lower pH (≤7.6) with or without urea, and a citrate buffer had no effect. However, a Tris-HCl buffer at pH 9.5 was effective at least for eNOS, and an addition of 5% urea to it enhanced this effect. For both bNOS and eNOS immunostaining, the most effective method was to preincubate sections in an aqueous solution of urea (6M) for 4 to 12hr prior to immunostaining. The sections treated with 6M urea solution showed strong immunostaining reactivity for bNOS in neurons dispersively distributed in the cerebral cortex and in nuclei of the brain stem. With the same pretreatment of sections, eNOS immunoreactivity was also clearly detected on the wall of small blood vessels and capillaries in the parenchyma of the brain.
