Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences.
Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences.
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细胞和组织切片的联合免疫和非放射性杂交细胞化学：固定、酶预处理和色原选择对抗原和 DNA 序列检测的影响。
DOI：
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发表时间：
1989
影响因子：
3.2
通讯作者：
C. Meijer
中科院分区：
文献类型：
--
作者：
H. Mullink;J. Walboomers;T. Tadema;D. Jansen;C. Meijer
结合DNA原位杂交，使用生物素标记的DNA探针，免疫组织化学的条件进行了研究，冷冻切片，细胞学制剂，石蜡切片。我们发现，冷冻切片和细胞学制备物适合于在丙酮、甲醇、乙醇或Carnoy中固定后的靶DNA原位杂交，而无需进一步的蛋白酶预处理。丙酮也非常适用于细胞表面或细胞骨架抗原的免疫染色。因此，我们进行了联合免疫酶和原位杂交染色使用这种固定剂。当免疫过氧化物酶染色与二氨基联苯胺/H2 O2，然后直接通过原位杂交得到最好的结果。除了免疫过氧化物酶，碱性磷酸酶-抗碱性磷酸酶（APAAP）染色与萘酚ASBI磷酸盐和新品红作为底物可以使用。在大多数情况下，使用氮蓝四唑和5-溴-4-氯-3-吲哚基磷酸盐作为底物的链霉亲和素-生物素化多聚碱性磷酸酶方法检测生物素化杂合体是优选的。在以下测试模型上研究双重染色：（a）冷冻扁桃体切片：细胞表面抗原（pan T）和核糖体DNA;（B）冷冻生殖器湿疣切片：细胞角蛋白和人乳头瘤病毒6 + 11型（HPV-6/11）DNA;（c）CaSKi细胞：细胞角蛋白和HPV-16 DNA;（d）感染的胎儿肺成纤维细胞：波形蛋白和巨细胞病毒（CMV）DNA。一个适应的程序遵循常规甲醛固定和石蜡包埋的湿疣组织。HPV-6/11 DNA原位杂交可与免疫过氧化物酶染色相结合。然而，在该模型中，靶DNA的可及性必须在免疫染色后和开始原位杂交前通过酶处理来改善。
Conditions for combination of DNA in situ hybridization, using biotinylated DNA probes, with immunohistochemistry were investigated on cryostat sections, cytological preparations, and paraffin sections. We found that cryostat sections and cytological preparations are suitable for in situ hybridization of target DNA after fixation in acetone, methanol, ethanol, or Carnoy without further proteinase pretreatment. Acetone is also very suitable for immunostaining of cell surface or cytoskeleton antigens. We therefore performed combined immunoenzyme and in situ hybridization staining using this fixative. The best results were obtained when immunoperoxidase staining with diaminobenzidine/H2O2 was followed directly by in situ hybridization. In addition to immunoperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) staining with naphthol ASBI phosphate and New Fuchsin as a substrate could be used. In most instances, detection of the biotinylated hybrid with a streptavidin-biotinylated polyalkaline phosphatase method using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as the substrate was preferable. The double stainings were studied on the following test models: (a) frozen tonsil sections: cell surface antigens (pan T) and ribosomal DNA; (b) frozen genital condyloma sections; cytokeratins and human papillomavirus type 6 + 11 (HPV-6/11) DNA; (c) CaSKi cells: cytokeratins and HPV-16 DNA; (d) infected fetal lung fibroblasts: vimentin and cytomegalovirus (CMV) DNA. An adapted procedure was followed on routinely formaldehye-fixed and paraffin-embedded condyloma tissue. Immunoperoxidase staining for papilloma virus capsid antigen could be combined with DNA in situ hybridization with HPV-6/11 DNA. In this model, however, the accessibility of the target DNA had to be improved by enzyme treatment after the immunostaining and before starting the in situ hybridization.
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在大鼠垂体的同一组织切片中结合免疫细胞化学和原位杂交。
DOI：
10.1177/34.1.3510246
发表时间：
1986
期刊：
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
影响因子：
--
作者：
Shivers,BD;Harlan,RE;Pfaff,DW;Schachter,BS
通讯作者：
Schachter,BS
DOI：
10.1073/pnas.78.11.6633
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
LANGER, PR;WALDROP, AA;WARD, DC
通讯作者：
WARD, DC
早期宫颈肿瘤中 HPV 16 DNA 序列的原位杂交分析。
DOI：
--
发表时间：
1986
期刊：
The American journal of pathology
影响因子：
--
作者：
Crum,CP;Nagai,N;Levine,RU;Silverstein,S
通讯作者：
Silverstein,S
DOI：
10.1073/pnas.80.13.4045
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
LEARY, JJ;BRIGATI, DJ;WARD, DC
通讯作者：
WARD, DC