Journal of Histochemistry & Cytochemistry

Loos, Chris M. van der

doi: 10.1369/jhc.2007.950170pmid: 18158282

Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, because of the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit–mouse, goat–mouse, mouse–mouse, and rabbit–rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (diaminobenzidine tetrahydrochloride), alkaline phosphatase activity (Liquid Permanent Red), and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.

Zhu, Shimei; Barbe, Mary F.; Amin, Neilay; Rani, Shobha; Popoff, Steven N.; Safadi, Fayez F.; Litvin, Judith

doi: 10.1369/jhc.7A7321.2007pmid: 18040074

Periostin-like factor (PLF) and Periostin are alternatively spliced mRNAs. Our findings are the first to show similarities and differences between PLF and Periostin location using isoform-specific antibodies. The differences in when and where they are present during mouse embryogenesis suggest that they may have different functions. Using immunostaining techniques, we observed that PLF was highly expressed at 12.5 days postconception (dpc) in the intermediate and outer zones of most brain regions, spinal cord, cranial and spinal nerves, and chondrocytes in developing bone and in the heart wall. By 16.5 dpc, PLF was also present in ameloblasts and odontoblasts in developing teeth, and by 19.5 dpc, PLF was present at low levels only in vagal nerve bundles, discrete white matter bundles in the brain, and chondrocytes of developing ribs. Periostin, on the other hand, was absent at 12.5 dpc from dorsal spinal cord and from cranial and spinal nerves. By 16.5 dpc, Periostin was present in many spinal nerves, but absent thereafter, and at 19.5 dpc, Periostin was present in chondrocytes in developing bone but not in neural tissues. The different spatial and temporal location of PLF and Periostin in cartilage and bone cells suggests different roles for these proteins in endochondral bone formation. The early expression of PLF in brain differentiation zones and in developing axon bundles and nerves suggests that it may facilitate axon growth.

Goodpaster, Tracy; Legesse-Miller, Aster; Hameed, Meera R.; Aisner, Seena C.; Randolph-Habecker, Julie; Coller, Hilary A.

doi: 10.1369/jhc.7A7287.2007pmid: 18071065

Fibroblasts are critical for tissue homeostasis, and their inappropriate proliferation and activation can result in common and debilitating conditions including fibrosis and cancer. We currently have a poor understanding of the mechanisms that control the growth and activation of fibroblasts in vivo, in part because of a lack of suitable fibroblast markers. We have taken advantage of an antibody previously shown to stain stromal cells in frozen tissues (TE-7) and identified conditions in which it can be used to stain fibroblasts and myofibroblasts in the paraffin-embedded tissue samples routinely collected for pathological analysis. We show that this antibody recognizes growing and quiescent fibroblasts and myofibroblasts by immunohistochemistry, immunofluorescence, and ELISA assays. We also present its staining patterns in normal tissue samples and in breast tumors.

Hu, Jin-Jia; Ambrus, Andy; Fossum, Theresa W.; Miller, Matthew W.; Humphrey, Jay D.; Wilson, Emily

doi: 10.1369/jhc.7A7324.2007pmid: 18071063

Arteries undergo marked structural and functional changes in human andexperimental hypertension that generally involve smooth muscle cell (SMC)hypertrophy/hyperplasia as well as abnormal extracellular matrix turnover. Inthis study we examined time courses of changes in SMC activity and matrixprotein content in a novel mini-pig aortic coarctation model. Cell proliferationwas evaluated by immunostaining of Ki-67, apoptosis was assessed by TUNEL, andphenotypic changes were monitored by immunostaining three SMC contractilemarkers (caldesmon, calponin, and smoothelin). Changes in medial collagen andelastin were examined by picrosirius red and Verhoeff–van Giesonstaining, respectively. LabVIEW-based image analysis routines were developed toobjectively and efficiently quantify the (immuno)histochemical results. We foundthat significant cell proliferation and matrix production occurred in the earlystages of this coarctation model and then declined gradually; the SMCs alsotended to exhibit a less contractile phenotype following these cellular andextracellular changes. Specifically, different aspects of the phenotypic changesassociated with hypertension occurred at different rates: cell proliferation andcollagen production occurred early and peaked by 2 weeks, whereas changes incontractile protein expression continued to decrease over the entire 8-weekstudy period. Temporal changes found in this study emphasize the importance ofsimultaneously tracing time courses of SMC growth and differentiation as well asmatrix protein production and content. SMCs are multifunctional, and cautionmust be used to not overdefine phenotype. This manuscript contains onlinesupplemental material at http://www.jhc.org. Please visit this article online to viewthese materials.

Lutz, Barry; Dentinger, Claire; Sun, Lei; Nguyen, Lienchi; Zhang, Jingwu; Chmura, AJ; Allen, April; Chan, Selena; Knudsen, Beatrice

doi: 10.1369/jhc.7A7313.2007pmid: 18071064

Surface-enhanced Raman scattering (SERS) nanoparticles are emerging as a new approach for optical detection of biomolecules. In a model assay in formalin-fixed paraffin-embedded (FFPE) prostate tissue sections, we detect prostate-specific antigen (PSA) using antibody (Ab) conjugated to composite organic–inorganic nanoparticles (COINs), and we use identical staining protocols to compare COIN-Ab and Alexa–Ab conjugates in adjacent tissue sections. Spectral analysis illustrates the fundamental difference between fluorescence and Raman signatures and accurately extracts COIN probe signals from background autofluorescence. Probe signals are used to generate images of PSA expression on the tissue, and quality measures are presented to characterize the performance of the COIN assay in comparison to Alexa. Staining accuracy (ability to correctly identify PSA expression in epithelial cells) is somewhat less for COIN than Alexa, which is attributed to an elevated false negative rate of the COIN. However, COIN provided signal intensities comparable to Alexa, and good intra-, inter-, and lot-to-lot consistencies. Overall, COIN and Alexa detection reagents possess similar performance with FFPE tissues, supporting the further development of Raman probes for this application. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Rindler, Michael J.; Xu, Chong-feng; Gumper, Iwona; Cen, Chuan; Sonderegger, Peter; Neubert, Thomas A.

doi: 10.1369/jhc.7A7351.2007pmid: 18158283

Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of α cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.

Michalczyk, Agnes; Bastow, Edward; Greenough, Mark; Camakaris, James; Freestone, David; Taylor, Philip; Linder, Maria; Mercer, Julian; Ackland, Margaret L.

doi: 10.1369/jhc.7A7300.2008pmid: 18180385

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper (64Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.

Park, Sungmi; Harrison-Bernard, Lisa M.

doi: 10.1369/jhc.2007.950220pmid: 18180383

The present study was performed to determine the influence of absence of angiotensin type 1A (AT1A) and/or AT1B receptor feedback regulation of kidney neuronal nitric oxide synthase (nNOS) and renin protein expression. Kidneys were harvested from wildtype (WT), AT1A−/-, AT1B−/-, and AT1A−/-AT1B−/- mice and immunostained for nNOS and renin protein localization. AT1A−/- and AT1A−/-AT1B−/- kidneys demonstrated an increase in the percentage of glomeruli with nNOS-positive afferent and interlobular arterioles compared with WT mice. Density of vascular nNOS immunostaining was 20-fold higher in kidneys of AT1A−/- and AT1A−/-AT1B−/- compared with WT mice. Density of macula densa nNOS immunostaining was 7-fold higher in AT1A−/-AT1B−/- than in WT mice. Percent of glomeruli positive for juxtaglomerular (JG) cell renin was 3-fold higher, whereas the density of JG cell renin immunostaining was 15-fold higher in kidneys of AT1A−/- and AT1A−/-AT1B−/- compared with WT mice. Kidneys of AT1A−/- and AT1A−/-AT1B−/- mice displayed recruitment of renin protein expression along afferent and interlobular arterioles. Absence of AT1 receptor signaling resulted in enhanced nNOS protein expression in both microvascular and tubular structures. Enhanced NO generation may contribute to the reduced renal vascular tone and blood pressure observed with blockade of the renin–angiotensin system.

Klinck, Rasmus; Serup, Palle; Madsen, Ole D.; Jørgensen, Mette C.

doi: 10.1369/jhc.7A7350.2008pmid: 18212389

The homeodomain transcription factor Nkx6-1 is essential for proper motor neuron development and development of insulin-producing pancreatic β-cells. Nkx6-1 is closely related to Nkx6-2 and Nkx6-3, and all three are expressed in the developing central nervous system and in the developing foregut. Immunohistochemical detection of protein expression is an important tool for description of the temporal differences in expression patterns. When several gene family members like the Nkx6 factors have overlapping or juxtaposed expression domains, there is an elevated risk of unrecognized cross-reactivity, and it is therefore crucial to determine the specificities of antibodies against such targets. In this study we have determined the epitope consensus sequences of four monoclonal antibodies against Nkx6-1 using SPOT membranes, and we refined the results by combined peptide recognition and blocking assays. We show that two of the monoclonal anti-Nkx6-1 antibodies specifically recognize Nkx6-1 and do not cross-react to Nkx6-2 and Nkx6-3. The other two monoclonal anti-Nkx6-1 antibodies are specific to Nkx6-1 in mice but do not recognize Nkx6-1 in chicken and human.