Validation of image analysis for enzyme histochemical and immunocytochemical staining

Immunocytochemical and enzyme histochemical analyses of cells and tissues are used to detect changes in the extent of injury and the expression of various molecules. Image analysis quantitation offers an easier, more efficient technique to evaluate these changes. We studied the application of image analysis for evaluating enzyme histochemistry and immunocytochemistry of cells and tissues as a way to assess stroke. Using brain sections, we compared investigator and computer-generated image analysis of 2,3,5-triphenyltetrazolium chloride stained cerebral infarcts in rats subjected to 2 h middle cerebral artery occlusion and 22 h re-perfusion. Both methods documented the infarct volumes with a comparison of means of less then 5%. This suggests no difference between computer- and hand-calculated values. Computer-generated analysis was easier and faster to use. Using endothelial cell monolayers, immunocytochemical staining of a time course of heat shock protein expression was compared to a grading system using fast red chromagen counterstained with hematoxylin. Results demonstrated greater ease and efficiency with computer-generated image analysis compared to other subjective systems of analysis. Image analysis is more useful for detecting small differences in staining, especially when using 3,3-diaminobenzidine as a chromagen. Investigator bias is also reduced using this system. Our comparisons validate the use of this versatile technology to assess more easily both cell and tissues in stroke research.     

Acute connexin43 temporal and spatial expression in response to ischemic stroke

Connexin43 (Cx43) gap junctions expressed in astrocytes can significantly impact neuronal survival in stroke. However, little is known regarding Cx43 spatial and temporal expression during the initial stages of brain ischemia. Using immunohistochemistry and Western blot analysis, we examined Cx43 spatial and temporal expression as a function of neuronal injury within the first 24 h after permanent middle cerebral artery occlusion (pMCAO). Western blot analysis showed a significant increase in Cx43 protein expression in the core ischemic area at 2 and 3 h after pMCAO. However, after 6 h of pMCAO Cx43 levels were significantly reduced. This reduction was due to cell death and concomitant Cx43 degradation in the expanding focal ischemic region, while the peri-infarct zone revealed intense Cx43 staining. The neuronal cell-death marker Fluoro-Jade C labeled injured neurons faintly at 1 h post-pMCAO with a time-dependent increase in both intensity and size of punctate staining. In addition, decreased microtubule-associated protein 2 (MAP2) immunoreactivity and thionin staining similarly indicated cell damage beginning at 1 h after pMCAO. Taken together, Cx43 expression is sensitive to neuronal injury and can be detected as early as 2 h post-pMCAO. These findings underscore Cx43 gap junction as a potential early target for therapeutic intervention in ischemic stroke.     

Western印迹中蛋白质定量分析的替代方法

Western印迹广泛采用显像密度计法对特异性蛋白质表达进行定量,但是仍然存在敏感性的问题尚未解决。同时,昂贵的试剂与复杂的设备限制了其应用。建立了一种改良定量检测法,使用碱性磷酸酶标记的第二抗体,以萘酚磷酸盐和快红作反应底物。吸附膜上的终末显色用有机溶剂洗脱,通过测定光吸收值定量。结果显示该法更简便、快速、经济而不失其敏感性。     

Computerized image analysis as a tool to quantify infiltrating leukocytes: a comparison between high- and low-magnification images.

The purpose of the present study was to establish a rapid and reproducible method for quantification of tissue-infiltrating leukocytes using computerized image analysis. To achieve this, the staining procedure, the image acquisition, and the image analysis method were optimized. Because of the adaptive features of the human eye, computerized image analysis is more sensitive to variations in staining compared with manual image analysis. To minimize variations in staining, an automated immunostainer was used. With a digital scanner camera, low-magnification images could be sampled at high resolution, thus making it possible to analyze larger tissue sections. Image analysis was performed by color thresholding of the digital images based on values of hue, saturation, and intensity color mode, which we consider superior to the red, green, and blue color mode for analysis of most histological stains. To evaluate the method, we compared computerized analysis of images with a x100 or a x12.5 magnification to assess leukocytes infiltrating rat brain tumors after peripheral immunizations with tumor cells genetically modified to express rat interferon-gamma (IFN-gamma) or medium controls. The results generated by both methods correlated well and did not show any significant differences. The method allows efficient and reproducible processing of large tissue sections that is less time-consuming than conventional methods and can be performed with standard equipment and software.(J Histochem Cytochem 49:1073-1079, 2001)     

Proliferating cell nuclear antigen in normal urothelium and urothelial lesions of the urinary bladder: a quantitative assessment using a true color image analysis system

To evaluate proliferating cell nuclear antigen (PCNA) staining for assessing proliferative activity in routine pathology specimens of urinary bladder, the bladder carcinoma cell line J82 and a total of 122 specimens of normal bladder and urothelial lesions were stained with the antibody clone PC10 against proliferating cell nuclear antigen. In in vitro plateau cultures the proportion of PCNA-positive cells exceeded that of Ki-67-positive cells, and only very few cells were negative. In formalin-fixed tissues, the PCNA staining pattern, which should be confined to replicon units in the nucleus, was optimized by 1 h postfixation in an organic solvent (methacarn). Sections showed positive nuclear staining confined to basal and some suprabasal cells in normal urothelium and grade 1 dysplasias, but more generalized nuclear staining in all other neoplastic lesions. In addition, stromal cells adjacent to invasive tumors showed nuclear positivity in some instances. Using quantitative true color image analysis of sections counterstained with hemalum, the degree of brown staining of the PCNA reaction product is contrasted with the blue staining of the nuclear area. With this method low contrast specific staining not appreciated optically can be reliably detected. Image analysis data confirmed observations made on noncounterstained sections and showed significant differences between grade 1 and 2 dysplasias as well as between grade 1 dysplasia and all grades of papillary tumor. Furthermore, a significant difference in PCNA staining indices was found between grade 1 and 3 bladder carcinomas. The results indicate that PCNA staining using the PC10 antibody is not confined to the proliferative fraction of neoplastic urothelium. In contrast with data from normal tissue and malignant hematological neoplasms, the amount of PCNA is regulated differently in urothelial neoplasms, emphasizing the biological differences between the following two sets: mild dysplasia and moderate dysplasia; mild dysplasia and papillary carcinomas. The use of image analysis to standardize the detection process after controlled staining conditions is advisable in order to provide reliable data. Supported by the DFG project: Knuechel/Urothelcarcinom 263     

Upregulation of EMMPRIN after permanent focal cerebral ischemia

Elevated activities of matrix metalloproteinases (MMPs) following ischemic stroke have been shown to mediate ischemic injury as well as neurovascular remodeling. The extracellular MMP inducer (EMMPRIN) is a 58-kDa cell surface glycoprotein, which has been known to play a key regulatory role for MMP activities. The roles of EMMPRIN in stroke injury are not clearly understood. In this study, we investigated changes of EMMPRIN in a mouse model of permanent focal cerebral ischemia, and examined potential association between EMMPRIN and MMP-9 expression. Adult male CD-1 mice were subjected to permanent focal ischemia by intraluminal occlusion of the left middle cerebral artery (MCAO) under anesthesia. EMMPRIN expression was markedly upregulated in the peri-infarct area at 2-7 days after ischemia compared to the contralateral non-ischemic hemisphere by Western blot analysis. Immunofluorescent double staining demonstrated that EMMPRIN signals co-localized with vwF-positive endothelial cells and GFAP-positive peri-vascular astrocytes. In contrast, EMMPRIN signal did not co-localize with NeuN-positive neurons, or MPO-positive neutrophils. Dual fluorescent staining revealed that EMMPRIN co-localized with MMP-9. Our data also demonstrated that increased EMMPRIN expression correlated with increased MMP-9 levels in a temporal manner. In summary, we report for the first time that EMMPRIN expression was significantly increased in a mouse model of permanent focal cerebral ischemia. The spatial and temporal association between increased EMMPRIN expression and elevated MMP-9 levels suggest that EMMPRIN may modulate MMP-9 activity, and participate in neurovascular remodeling after ischemic stroke.     

COX-2和IL-1在甲状腺乳头状癌组织中的表达

目的探讨COX-2和IL-1在甲状腺乳头状癌组织中的表达及其在肿瘤的发生和发展中的作用。方法收集武汉大学人民医院和武汉大学中南医院病理科2000-2006年手术切除及活检的甲状腺乳头状癌标本共40例,另取癌周围组织5例作对照。采用免疫组织化学方法观察各组组织内COX-2和IL-1的表达。利用HPIAS-2000图像分析系统测定COX-2和IL-1在癌及癌旁组中表达的平均光密度和平均阳性面积率。结果甲状腺乳头状癌组织中COX-2和IL-1呈高表达;癌旁组织中COX-2和IL-1呈低表达。图像分析结果显示两组间差异有显著性意义（P〈0.01）。结论IL-1可能通过诱导COX-2的表达,在促进肿瘤的发生和发展中起作用。     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed,paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

正常与脑缺血大鼠的脑皮质蛋白质差异分析鉴定

Wistar大鼠随机分为正常组和模型组,采用改进的线栓法制备模型,在规定的时间点快速断头取脑,分离脑皮质组织,提取蛋白质后双向电泳展示,以ImageMaster 2D Elite v301软件对2_DE图谱进行差异表达分析,目标蛋白点用基质辅助激光解析电离质谱测定肽质量指纹图进行鉴定。线粒体应激70蛋白前体、血小板活化因子乙酰基水解酶Ibβ亚单位、ADP核糖基化因子蛋白3、电压依赖性阴离子选择通道蛋白1、泛素C末端水解酶同工酶L1、突触结合蛋白等11个蛋白在模型6h组表达上调,谷胱甘肽S-转移酶omega 1、 谷胱甘肽S-转移酶P、Cu-Zn超氧化物歧化酶、 ATP合酶D链、G蛋白β亚单位1、微管蛋白β链15、苹果酸脱氢酶等15个蛋白在模型6h组表达上调。胆绿素还原酶B、细胞因子A4前体为模型组新出现点,腺苷酸激酶同工酶1在模型组消失,Thiore doxin peroxidase 1在模型组分为2个点。以双向电泳技术得到分辨率较好的电泳图谱,并初步鉴定脑缺血后差异表达蛋白,为深入研究缺血性脑损伤病理机制奠定了基础。     

Enhanced protection by melatonin and meloxicam combination in a middle cerebral artery occlusion model of acute ischemic stroke in rat

Mixed efficacy of neuroprotective drugs in clinical trials has led to the emergence of the approach of combination therapy in stroke. The present study was carried out to investigate the effect of the combination of melatonin (potent antioxidant) and meloxicam (preferential inhibitor of cyclooxygenase-2 enzyme) against a middle cerebral artery occlusion model of stroke in rats. Male Wistar rats in the weight range of 250-300 g were used. Rats were anesthetized using chloral hydrate (400 mg/kg i.p) and subjected to 2 h of transient middle cerebral artery occlusion. Melatonin was administered at a dose of 20 mg/kg i.p. four times: at the time of middle cerebral artery occlusion, 1.5 h after middle cerebral artery occlusion, at the time of reperfusion, and 1 h after reperfusion. Meloxicam (2.5 mg/kg) was administered 4 h after middle cerebral artery occlusion. Motor performance tests (grip test, foot fault test, rotarod performance test, spontaneous locomotor activity), markers of oxidative stress, and triphenyltetrazolium chloride staining were carried out 24 h after middle cerebral artery occlusion. A vehicle-treated group was run in parallel. It was observed that melatonin treatment improved the motor performance and significantly attenuated the levels of malondialdehyde (MDA) as compared with the middle cerebral artery occluded group. Meloxicam treatment at the dose used neither showed significant improvement on the motor performance nor decreased the levels of MDA significantly as compared with the middle cerebral artery occluded group. However, when the combination of the two drugs was used, better protection was observed as was evident by the significant decrease in the percent foot fault errors, the increase in the time spent on the rotarod, and the increase in the six-point neurological score and grip test score. There was also a significant decrease in the levels of MDA in the combination group. The results of the present study demonstrate that enhanced protection is observed with the use of a combination of melatonin plus meloxicam in the middle cerebral artery occlusion model of acute ischemic stroke in rats.     

Immunohistochemical analysis of Bin1/Amphiphysin II in human tissues: diverse sites of nuclear expression and losses in prostate cancer

The Bin1/Amphiphysin II gene encodes at least seven alternately spliced adapter proteins that have been implicated in membrane dynamics and nuclear processes. Nuclear localized Bin1 polypeptides have tumor suppressor and proapoptotic activities, suggesting that Bin1 may suppress cancer in tissues where nuclear expression may occur. One question is the extent to which human tissues express nuclear Bin1 isoforms. A secondary issue has been the need for a specific antibody that can detect all the splice isoforms expressed by the human, mouse, and rat Bin1 genes. Using a novel mouse monoclonal antibody with these characteristics, we performed an immunohistochemical analysis of Bin1 expression in a panel of normal human tissues. We also compared the expression profile of Bin1 in normal or malignant tissues derived from human prostate, where Bin1 is a candidate tumor suppressor gene. In brain, a distinct nuclear staining pattern overlapped with a cytosolic staining pattern present in certain layers of the cerebral cortex and cerebellum. Bone marrow cells displayed mainly nuclear localization whereas peripheral lymphoid cells exhibited mainly cytosolic localization. In several epithelial tissues, nuclear or nucleocytosolic staining patterns were displayed by basal cells in skin, breast, or prostate, whereas cytosolic or plasma membrane-associated staining patterns were noted in gastrointestinal cells. Interestingly, a striking gradient of expression was observed in gastrointestinal epithelia, particularly in the large intestine, with the strongest staining displayed by cells destined to undergo apoptosis at the villus tip. In prostate, Bin1 staining was frequently absent in cases of primary prostate adenocarcinoma. This study used a novel reagent to document the extent of expression of nuclear Bin1 isoforms, which exhibit cancer suppression and proapoptotic activity in human cells.     

Localization of alpha 2u-globulin within protein droplets of male rat kidney: immunohistochemistry using perfusion-fixed, GMA-embedded tissue sections

We investigated the light microscopic subcellular localization and quantitation of alpha 2u-globulin (alpha 2uG) in male rat kidney after 2,2,4-trimethylpentane exposure using a monoclonal antibody to alpha 2uG. Slices of perfusion-fixed kidney were cold-processed in glycolmethacrylate and the antigen localized after an avidin-biotin-horseradish peroxidase procedure with Hanker-Yates reagent as the chromagen. Light microscopic examination revealed resolution comparable to low-magnification electron microscopy, with excellent morphological detail of tissue architecture, including subcellular localization of alpha 2uG within lysosomes of P2 segment cells of the proximal tubule epithelium. The anatomical relationship between alpha 2uG and TMP-induced protein droplets observed after staining with Lee's methylene blue-basic fuchsin was studied using serial sections. Image analysis of selected P2 segments in treated and control rats revealed a high correlation between subcellular localization of alpha 2uG and protein droplet deposition in the cytoplasm of P2 segment cells of the proximal tubule epithelium. Quantitative morphometry of alpha 2uG-stained proximal tubule epithelium 72 hr after treatment with 50 mg/kg 2,2,4-trimethylpentane p.o. demonstrated a 1.5- to 2-fold increase in staining area of tubules from treated rats compared with controls. Similar increases of Lee's methylene blue-basic fuchsin-stained protein droplets were also observed, but quantitative morphometry of the protein droplets was technically more difficult owing to a lower staining contrast between droplets and the surrounding cytoplasm. This immunohistochemical procedure provides a valuable technique for further studies on the pathological role of alpha 2uG in protein droplet nephropathy of male rats induced by many environmental chemicals, and demonstrates the value of cold glycolmethacrylate processing to improve morphological detail.     

PA-MSHA引起注射局部急性炎症及白细胞介素17、肿瘤坏死因子α和Toll样受体4的表达改变

为研究皮下注射铜绿假单胞菌甘露糖敏感血凝菌毛株(Pseudomonas aeruginosa mannose sensitive hemagglutinin,PA-MSHA)注射液后局部组织的病理学改变及白细胞介素17(interleukin 17,IL-17)、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)和Toll样受体4(Toll-like receptor 4,TLR4)表达情况,将30只小鼠随机分为两组,分别皮下注射PA-MSHA和生理盐水,在注射后6、12、24、48及72h取注射局部组织,苏木精-伊红(hematoxylin-eosin,HE)染色后观察病理形态改变,并用免疫组化染色和图像分析检测IL-17、TNF-α和TLR4表达水平。结果显示,小鼠皮下注射PA-MSHA后,在给药部位引起以中性粒细胞浸润为主的急性炎症;局部炎症灶中IL-17、TNF-α和TLR4表达在注射后6h升高,24h达到高峰,然后逐渐减低,至72h实验组与对照组无差异;3种炎症因子的表达与炎症反应程度相平行。结果表明,皮下注射PA-MSHA能引起局部组织急性炎症反应,且能改变局部免疫状态。     

Examination of 2-DE in the Human Proteome Organisation Brain Proteome Project pilot studies with the new RAIN gel matching technique

The Human Proteome Organisation (HUPO) Brain Proteome Project (BPP) pilot studies have generated over 200 2-D gels from eight participating laboratories. This data includes 67 single-channel and 60 DIGE gels comparing 30 whole frozen C57/BL6 female mouse brains, ten each at embryonic day 16, postnatal day 7 (juvenile) and postnatal day 54-56 (adult); and ten single-channel and three DIGE gels comparing human epilepsy surgery of the temporal front lobe with a corresponding post-mortem specimen. The samples were generated centrally and distributed to the participating laboratories, but otherwise no restrictions were placed on sample preparation, running and staining protocols, nor on the 2-D gel analysis packages used. Spots were characterised by MS and the annotated gel images published on a ProteinScape web server. In order to examine the resultant differential expression and protein identifications, we have reprocessed a large subset of the gels using the newly developed RAIN (Robust Automated Image Normalisation) 2-D gel matching algorithm. Traditional approaches use symbolic representation of spots at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in spot modelling and matching. With RAIN, image intensity distributions, rather than selected features, are used, where smooth geometric deformation and expression bias are modelled using multi-resolution image registration and bias-field correction. The method includes a new approach of volume-invariant warping which ensures the volume of protein expression under transformation is preserved. An image-based statistical expression analysis phase is then proposed, where small insignificant expression changes over one gel pair can be revealed when reinforced by the same consistent changes in others. Results of the proposed method as applied to the HUPO BPP data show significant intra-laboratory improvements in matching accuracy over a previous state-of-the-art technique, Multi-resolution Image Registration (MIR), and the commercial Progenesis PG240 package.     

Counting pollen grains using readily available, free image processing and analysis software

Background and Aims

Although many methods exist for quantifying the number of pollen grains in a sample, there are few standard methods that are user-friendly, inexpensive and reliable. The present contribution describes a new method of counting pollen using readily available, free image processing and analysis software.

Methods

Pollen was collected from anthers of two species, Carduus acanthoides and C. nutans (Asteraceae), then illuminated on slides and digitally photographed through a stereomicroscope. Using ImageJ (NIH), these digital images were processed to remove noise and sharpen individual pollen grains, then analysed to obtain a reliable total count of the number of grains present in the image. A macro was developed to analyse multiple images together. To assess the accuracy and consistency of pollen counting by ImageJ analysis, counts were compared with those made by the human eye.

Key Results and Conclusions

Image analysis produced pollen counts in 60 s or less per image, considerably faster than counting with the human eye (5–68 min). In addition, counts produced with the ImageJ procedure were similar to those obtained by eye. Because count parameters are adjustable, this image analysis protocol may be used for many other plant species. Thus, the method provides a quick, inexpensive and reliable solution to counting pollen from digital images, not only reducing the chance of error but also substantially lowering labour requirements.     

Expression of Placenta growth factor (PlGF) in non-Small cell Lung cancer (NSCLC) and the clinical and prognostic significance

Background

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. Over-expression of PlGF is known to be associated with pathological angiogenesis. This study examined PlGF expression at protein and message levels in non-small cell lung cancer (NSCLC), in which no reports on the significance of PlGF expression is available to date.

Patients and methods

We used immunohistochemistry to assess the PlGF protein and correlated PlGF with microvessel density (MVD), as well as clinical outcome in patients with NSCLC tumours (n = 91). In addition, we applied a real time quantitative PCR assay using SYBR Green chemistry to measure PlGF mRNA in normal lung tissues and NSCLC tumours.

Results

PlGF was positively stained mainly in cytoplasm of lung cancer cells. High level staining of PlGF was found in 38.5% NSCLC patients. A high level of MVD in NSCLC was found in 42.9% of cases. Tumours with high level and low level PlGF staining had a significantly different MVD (26.69 vs. 20.79, respectively, p = 0.003). Using both univariate and multivariate analyses, PlGF was found to be an independent prognostic factor. Real time PCR analysis revealed that PlGF mRNA was higher in the cancer tissue than normal tissue (0.95 ± 0.19 vs. 0.57 ± 0.24; p < 0.005) and that PlGF mRNA was significant higher in III-IV stage patients than in I-II stage patients (1.03 ± 0.20 vs. 0.80 ± 0.17; p = 0.011).

Conclusion

PlGF expression is significantly more in NSCLC tumour tissues than in matched normal tissues. It has a significant positive association with MVD and is an independent factor for NSCLC patients. PlGF may have a pivotal role in NSCLC development and disease progression.     

Time-dependent contribution of non neuronal cells to BDNF production after ischemic stroke in rats

Although brain-derived neurotrophic factor (BDNF) plays a central role in recovery after cerebral ischemia, little is known about cells involved in BDNF production after stroke. The present study testes the hypothesis that neurons are not the unique source of neosynthesized BDNF after stroke and that non neuronal-BDNF producing cells differ according to the delay after stroke induction. For this purpose, cellular localization of BDNF and BDNF content of each hemisphere were analysed in parallel before and after (4h, 24h and 8d) ischemic stroke in rats. Stroke of different severities was induced by embolization of the brain with variable number of calibrated microspheres allowing us to explore the association between BDNF production and neuronal death severity. The main results are that (a) unilateral stroke increased BDNF production in both hemispheres with a more intense and long-lasting effect in the lesioned hemisphere, (b) BDNF levels either of the lesioned or unlesioned hemispheres were not inversely correlated to neuronal death severity whatever the delay after stroke onset, (c) in the unlesioned hemisphere, stroke resulted in increased BDNF staining in neurons and ependymal cells (at 4h and 24h), (d) in the lesioned hemisphere, beside neurons and ependymal cells, microglial cells (at 24h), endothelial cells of cerebral arterioles (at 4h and 24h) and astrocytes (at 8d) exhibited a robust BDNF staining as well. Taken together, overall data suggest that non neuronal cells are able to produce substantial amount of BDNF after ischemic stroke and that more attention should be given to these cells in the design of strategies aimed at improving stroke recovery through BDNF-related mechanisms.