Kobayashi S, Ohashi T, Uchida T, Nakao K, Imura H, Yanaihara N, Verhofstad A A
Arch Histol Jpn. 1984 Aug;47(3):319-36. doi: 10.1679/aohc.47.319.
The co-existence of catecholamines with opioid peptides in adult hamster adrenal chromaffin cells was studied by Sternberger's peroxidase-antiperoxidase (PAP) method. Paraffin embedded serial sections (3-4 microns thick) of adrenal glands perfusion-fixed with either Bouin's fluid or 4% paraformaldehyde solution were immunostained using anti-adrenaline (AP16-2), -noradrenaline (NAP1-18), -Met-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-Arg-Gly-Leu: R-0171, NE2-206) and -Met-enkephalin-Arg6-Phe7 (Met-Enk-Arg-Phe: AP3-311) sera. Selective and specific immunostaining for adrenaline and noradrenaline was obtained in tissues fixed in 4% paraformaldehyde solution. Bouin's fluid gave negative or unsatisfactory results. Immunostaining with two kinds of Met-Enk-Arg-Gly-Leu (R-0171, NE2-206) and one kind of Met-Enk-Arg-Phe sera (AP3-311) was dependent upon the method of fixation used. The immunostaining in the Bouin-fixed tissues was stronger than that in the 4% paraformaldehyde-fixed ones. Secretory granules in chromaffin cells were responsible for both adrenaline- and noradrenaline-like and Met-Enk-Arg-Gly-Leu- and Met-Enk-Arg-Phe-like immunoreactivities. All adrenaline-storing (A) and noradrenaline-storing (NA) cells showed intense Met-Enk-Arg-Gly-Leu- and Met-Enk-Arg-Phe-like immunoreactivities. In the Bouin-fixed adrenal medulla, immunostaining of A cells with the two kinds of anti-Met-Enk-Arg-Gly-Leu sera (R-0171, NE2-206) was deeper than that of the NA cells. Immunostaining of A cells with the anti-Met-Enk-Arg-Phe serum (AP3-311) was apparently of the same intensity as that of NA cells. In 4% paraformaldehyde-fixed tissues, no correlation was seen between the distribution of A and NA cells and that of intensely and slightly Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe immunopositive cells. The "masking" of immunoreactive residues in chromaffin granules by an aldehyde-noradrenaline complex may be one of the major causes of the decreased opioid peptide-like immunoreactivity in the 4% paraformaldehyde-fixed adrenal medulla.
采用Sternberger过氧化物酶-抗过氧化物酶(PAP)法研究成年仓鼠肾上腺嗜铬细胞中儿茶酚胺与阿片肽的共存情况。用Bouin氏液或4%多聚甲醛溶液灌注固定肾上腺,制成石蜡包埋的连续切片(厚3 - 4微米),使用抗肾上腺素(AP16 - 2)、抗去甲肾上腺素(NAP1 - 18)、抗甲硫氨酸脑啡肽-精氨酸6 - 甘氨酸7 - 亮氨酸8(甲硫氨酸脑啡肽-精氨酸-甘氨酸-亮氨酸:R - 0171,NE2 - 206)和抗甲硫氨酸脑啡肽-精氨酸6 - 苯丙氨酸7(甲硫氨酸脑啡肽-精氨酸-苯丙氨酸:AP3 - 311)血清进行免疫染色。在4%多聚甲醛溶液固定的组织中获得了对肾上腺素和去甲肾上腺素的选择性和特异性免疫染色。Bouin氏液染色结果为阴性或不理想。用两种甲硫氨酸脑啡肽-精氨酸-甘氨酸-亮氨酸(R - 0171,NE2 - 206)和一种甲硫氨酸脑啡肽-精氨酸-苯丙氨酸血清(AP3 - 311)进行免疫染色取决于所用的固定方法。Bouin氏液固定的组织中的免疫染色比4%多聚甲醛固定的组织中的更强。嗜铬细胞中的分泌颗粒负责肾上腺素样、去甲肾上腺素样以及甲硫氨酸脑啡肽-精氨酸-甘氨酸-亮氨酸样和甲硫氨酸脑啡肽-精氨酸-苯丙氨酸样免疫反应性。所有储存肾上腺素(A)和储存去甲肾上腺素(NA)的细胞都显示出强烈的甲硫氨酸脑啡肽-精氨酸-甘氨酸-亮氨酸样和甲硫氨酸脑啡肽-精氨酸-苯丙氨酸样免疫反应性。在Bouin氏液固定的肾上腺髓质中,用两种抗甲硫氨酸脑啡肽-精氨酸-甘氨酸-亮氨酸血清(R - 0171,NE2 - 206)对A细胞的免疫染色比NA细胞更深。用抗甲硫氨酸脑啡肽-精氨酸-苯丙氨酸血清(AP3 - 311)对A细胞的免疫染色强度显然与NA细胞相同。在4%多聚甲醛固定的组织中,A和NA细胞的分布与强烈和轻微甲硫氨酸脑啡肽-精氨酸-甘氨酸-亮氨酸及甲硫氨酸脑啡肽-精氨酸-苯丙氨酸免疫阳性细胞的分布之间没有相关性。嗜铬颗粒中免疫反应性残基被醛-去甲肾上腺素复合物“掩盖”可能是4%多聚甲醛固定的肾上腺髓质中阿片肽样免疫反应性降低的主要原因之一。