Xie Jin Han, Hollande Frédéric, Haynes John M, Pouton Colin W, Ventura Sabatino
Prostate Research Cooperative, Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
J Pharmacol Toxicol Methods. 2010 Mar-Apr;61(2):98-101. doi: 10.1016/j.vascn.2009.11.001. Epub 2009 Nov 27.
The use of antibodies to proliferating cell nuclear antigen (PCNA) for the immunohistochemical detection of proliferating cells has been limited in frozen tissue sections because of its temperature dependence and instability in formaldehyde. In this study various protocols for the immunohistochemical staining of PCNA in frozen mouse prostate tissue sections were tested in order to identify optimal conditions.
Fresh prostate tissues from 8 week old mice were frozen in liquid nitrogen or fixed with formaldehyde or paraformaldehyde before freezing with liquid nitrogen. Frozen tissues were then cut in a cryostat and unfixed sections were fixed by dipping slides with sections into fixative. Slide-mounted tissue sections were permeabilized with Triton X-100 before incubating overnight in primary antibody to PCNA diluted to different concentrations in different diluting media at room temperature or 4 degrees C. Secondary antibody was applied in the same medium as the primary. 19 different experimental protocols were examined.
Only one protocol showed strong positive immunostaining for PCNA. PCNA-immunopositive cells were observed in greater abundance in the stromal layer.
Paraformaldehyde fixation with Triton X-100 permeabilization without any blocking protocol produced the strongest nuclear PCNA immunolabeling probably when cells are in S-phase of mitosis which indicates the feasibility of PCNA immunoflourescence staining on frozen tissues.
用于增殖细胞核抗原（PCNA）免疫组织化学检测增殖细胞的抗体，在冷冻组织切片中的应用受到限制，因为其具有温度依赖性且在甲醛中不稳定。在本研究中，对冷冻小鼠前列腺组织切片中PCNA免疫组织化学染色的各种方案进行了测试，以确定最佳条件。
将8周龄小鼠的新鲜前列腺组织在液氮中冷冻，或在液氮冷冻前用甲醛或多聚甲醛固定。然后在低温恒温器中切割冷冻组织，将带有切片的载玻片浸入固定剂中对未固定的切片进行固定。在室温或4℃下，用Triton X-10进行通透处理后，将载有组织切片的载玻片在稀释于不同稀释介质中的不同浓度的PCNA一抗中孵育过夜。二抗与一抗使用相同的介质。共检查了19种不同的实验方案。
只有一种方案显示出对PCNA的强阳性免疫染色。在基质层中观察到更多的PCNA免疫阳性细胞。
在没有任何封闭方案的情况下，用Triton X-100通透处理的多聚甲醛固定，可能在细胞处于有丝分裂S期时产生最强的核PCNA免疫标记，这表明PCNA免疫荧光染色在冷冻组织上的可行性。
