Antigen retrieval on formaldehyde-fixed paraffin sections: its potential drawbacks and optimization for double immunostaining

It is well known that the major artifact induced by formaldehyde fixation is the masking of tissue antigens due to cross-linking of protein amino acid residues. Recently many antigen retrieval techniques have been devised to unmask the hidden antigen epitopes and recover immunoreactivity. In this study, some practical problems of two common unmasking techniques, i.e. heat-induced epitope retrieval and enzyme digestion have been reviewed in immunostaining of proliferating cell nuclear antigen (PCNA) on formaldehyde-fixed paraffin-embedded sections. As the heating conditions became more severe, false-positive staining and/or nonspecific background staining occurred. Based on the principle of protein inactivation/denaturation and the possible mechanisms of antigen retrieval, it has been suggested that the antigen retrieval itself can also denature proteins in tissues, just as many other protein inactivation processes. Thus, the total magnitude of protein conformational change caused by the overall unmasking procedure is in practice crucial. To prove this hypothesis and to overcome such undesirable drawbacks after antigen retrieval, a new combination technique of a mild heating condition (microwaved at 80°C for 15–20 min) and pepsin digestion was devised. This technique led to a strong specific immunoreactivity of PCNA, without any undesirable false positive or background staining. The procedure was also adapted for double immunostaining of PCNA together with -actin, bromodeoxyuridine, keratin, type IV collagen and vimentin.     

Multiplex chemiluminescence microscope imaging of P16INK4A and HPV DNA as biomarker of cervical neoplasia

Classification of cervical intraepithelial neoplasia (CIN) lesions in low-grade (CIN1) or high-grade (CIN2-3) ones is crucial for optimal patient management, but current histological diagnosis on bioptic samples is often hampered by inter-observer variability. To allow objective classification, we have exploited the peculiar characteristics of chemiluminescence detection, such as high sensitivity and easy quantification of the luminescence signal, to perform sequentially in the same tissue section both an immunohistochemical quantitative detection of p16^INK4A (a protein marker of high-grade CIN lesions) and an in situ hybridization for human papillomavirus (generally accepted as a necessary but insufficient cause of cervical carcinoma). Different label enzymes (alkaline phosphatase and horseradish peroxidase) were employed in order to avoid any interference between the two assays, and quantitative chemiluminescence image analysis was used to obtain objective evaluation of sample positivity. The multiplexed method allowed detection of two complementary biomarkers and provided discrimination between different lesions (non-neoplastic, low-grade and high-grade CIN). This assay might thus represent an accurate and objective diagnostic test providing important information for counseling, selection of therapy and follow up after surgical treatment.     

Immuno-laser capture microdissection of frozen prolactioma sections to prepare proteomic samples

Laser capture microdissection (LCM) technology combined with immunohistochemistry (immuno-LCM) is a valuable tool to obtain specific target cell populations and therefore this technique enables more accurate proteomic profile. In this study, we optimized the regular immuno-LCM technique to isolate and stain pure prolactin cells from either normal human pituitary (n = 6) or prolactioma (n = 11). Compared with the routine procedure, more intense and specific staining could be obtained when sections were pretreated with 0.2% Triton X-100 for 4 min. Interestingly, longer pretreatment (0.2% Triton X-100 for 10 min) or higher concentration (2% Triton X-100 for 4 and 10 min) greatly impaired labeling intensity and cell shape. Further scanning electron microscope study revealed that the component extracted from the cell surface by Triton X-100 was lipid. Using the optimized immuno-LCM technique, more pure prolactin cells could be isolated and prepared for further proteomic analysis. Taken together, we reported an optimized immuno-LCM technique that could effectively dissect pure target cells in different type pituitary adenomas for further proteomics analysis.     

稀土上转换发光材料标记抗体的制备及在免疫组化中的应用

采用微波辐射法合成了具有上转换发光特性的六方相纳米粒子NaGdF₄: Yb³⁺,Er³⁺(UCNPs), 其晶粒大小约为65 nm, 且粒子在980 nm的激发光下显示绿光(550 nm). 进一步在NaGdF₄: Yb³⁺,Er³⁺纳米晶的表面包覆了一层二氧化硅层, 进行氨基功能化后获得了表面共价结合氨基基团的粒径为70 nm的上转换发光纳米微球NaGdF₄: Yb³⁺,Er³⁺@SiO₂-NH₂(UCNPs@SiO₂-NH₂). 通过共价键将UCNPs@SiO₂-NH₂与多克隆抗体免疫球蛋白联接, 将标记后的多克隆抗体应用于传统的免疫组化检测子宫内膜腺细胞中基质金属蛋白酶组织抑制剂-4(TIMP-4)蛋白的表达. 结果表明, 微波合成的稀土上转换发光纳米材料形貌规则且粒径均一, 包覆硅壳后材料具有良好的分散性和水溶性, 荧光强度高且稳定, 在980 nm激发光下对生物组织无背景荧光, 可以很好地检测组织中蛋白质的表达.     

Inhibition of Salidroside on Salivary Gland Adenoid Cystic Carcinoma

To evaluate the role of salidroside on proliferation,apoptosis and invasiveness of salivary gland adenoid cystic carcinoma cells(SACC),immunocytochemical staining was employed to detect proliferating cell nuclear antigen(PCNA),caspase 3 and caspase 8 expression in SACC-2 cells.Modified Boyden chamber assay combined with laser confocal microscopy(LSCM) was used to evaluate the invasion and migration abilities of SACC-2 cells at different time point.Immunohistochemistry staining revealed that the expression of PCNA was significantly decreased(P0.01) after salidroside treatment.In contrast,salidroside treatment led to increased caspase 3 and caspase 8 in SACC-2 cells.Cell migration depth and number of cells that penetrated Boyden chamber were also decreased by salidroside.Salidroside potently inhibits the proliferation and simultaneously induces the apoptosis of SACC-2 cells.Migration and invasion of SACC-2 cells are also inhibited.Our data throw light on potential clinical application of salidroside to the patients with SACC.     

宫颈癌中EGFR蛋白表达及其临床病理意义

目的探讨表皮生长因子受体1（EGFR）蛋白表达在宫颈癌发生、发展中的临床病理意义。方法采用免疫组化EnVision法检测120例宫颈癌组织、49例高级别上皮内瘤变（CIN2~3）、40例低级别上皮内瘤变（CIN1）、37例正常宫颈组织中EGFR蛋白的表达,并进行比较分析。结果（1）正常宫颈、CIN1、CIN2~3、宫颈癌组织中EGFR蛋白表达阳性率分别为10.8%（4/37）、12.5%（5/40）、42.9%（21/49）、89.2%（107/120）。宫颈癌组织中EGFR蛋白表达阳性率显著高于CIN2~3、CIN1及正常宫颈组织（均P＜0.01）;EGFR蛋白在CIN2~3中表达显著高于CIN1和正常宫颈组织（均P＜0.01）;CIN1和正常宫颈组织之间EGFR蛋白表达阳性率无统计学差异（P＞0.05）。（2）肿瘤浸润深度更深的宫颈癌患者宫颈癌组织中EGFR蛋白表达阳性率高于浸润深度较浅的患者（P＜0.01）。结论EGFR在宫颈癌进展过程中起了重要作用,有望成为抗宫颈癌治疗的一个潜在生物学标志。     

Preparation and Application of Monoclonal Antibody Against hNDRG2

The full-length hNdrg2 cDNA-coded 357 amino acids was cloned and expressed in Escherichia coli strain DH5α as a 6× His-tagged protein. The purified 6× His-fusion protein was used to immunize mice for preparing monoclonal antibodies (mAb) against N-myc downstream-regulated gene 2 (Ndrg2). A hybridoma secreting a monoclonal antibody against Ndrg2 was obtained and named FMU-Ndrg2.3. Western blot analysis confirmed that this mAb is specific only to Ndrg2 but not to Ndrg1, Ndrg3, and Ndrg4-B. Some tissue distribution features of Ndrg2 proteins, such as thyroid, kidney, testis, prostate, and pancreas islets, were present by immunohistochemistry.     

Neuropilin-1与胃癌发生、发展及血管生成的关系

目的探讨胃癌组织中Neuropilin-1、VEGF的表达与胃癌生物学行为及血管生成的关系和意义.方法应用免疫组化ElivisionTM法检测正常胃黏膜、上皮内瘤变、胃癌组织中Neuropilin-1、VEGF、CD34的表达并测定MVD,回顾分析胃癌患者的临床病理资料.结果胃癌组织中Neuropilin-1、VEGF的表达及MVD高于正常胃黏膜和上皮内瘤变组织,且与胃癌淋巴结转移、浸润程度密切相关.Neuropilin-1、VEGF表达阳性组的MVD高于各自阴性组.两者表达呈正相关关系,联合表达时MVD增高.结论 Neuropilin-1、VEGF、血管生成参与了胃癌发生、浸润与转移;Neuropilin-1、VEGF参与了胃癌的血管生成;表达存在协同作用;联合表达时血管生成的效应增强.Neuropilin-1作为肿瘤抗血管治疗的靶点具有一定的价值.     

小鼠子宫腺肌病模型病灶中雌孕激素受体的表达及意义

目的探讨雌激素受体(ER)和孕激素受体(PR)在子宫腺肌病模型病灶中的表达及临床意义.方法用异体垂体移植法建立ICR小鼠子宫腺肌病模型,应用免疫组化SP法检测正常子宫内膜、模型子宫内膜及病灶异位子宫内膜中ER、PR的表达.结果垂体移植4个月和6个月后,均成功诱发子宫腺肌病模型,不同时间诱发模型的病灶大体结节数和HE染色评分之间比较,均无统计学差异(均P>0.05);建模6个月后模型病灶的结节显著大于建模4个月后(P<0.05).建模后4个月,模型子宫内膜和病灶异位子宫内膜以及正常子宫内膜中ER和PR表达的阳性率比较,均无统计学差异(均P>0.05);建模后6个月,3种子宫内膜中ER阳性率分别为55.07%、31.63%和24.74%;PR阳性率为48.40%、27.48%和22.70%;3者比较均有统计学差异(均P<0.05).结论子宫腺肌病的发生、发展可能与子宫内膜局部ER、PR的高表达相关.     

结直肠癌多层螺旋CT浸润征像与病理预后因子的关系

目的 探讨结直肠癌多层螺旋CT（MSCT）所见的浸润征象与病理预后因子的关系。方法 对32例结直肠癌患者,术前均行MSCT 水成像双期增强扫描,并用免疫组织化学方法检测手术病理标本中的p53、Ki67 阳性表达率及病理对照分析。结果 结直肠癌肠外浸润者p53、Ki67的阳性表达率分别为62.6％、74.5％,均高于肠壁肌层浸润者;区域淋巴结及远处转移者p53、Ki67的阳性表达率分别为65.8％、76.2％,均高于无淋巴结转移者（均P＜0.05）。结论 结直肠癌MSCT所见的浸润程度及区域淋巴结状态可作为临床评价结直肠癌的恶性度和预后的一个影像学指标,对术前判断结直肠癌术后复发转移潜能和预后有重要价值。