Am J Pathol. 2008 July; 173(1): 68–76.

Histological AnalysisSections (5 μm) of paraffin-embedded irradiated skin sections were cut and immunostained to identify neutrophils with rat anti-mouse neutrophil antibody (Serotec, Raleigh, NC) at 0.5 μg/ml and macrophages with rat anti-mouse Mac-3 (BD PharMingen, San Diego, CA) at 2.5 μg/ml as previously described.9,10 Briefly antigen-antibody complexes were detected using the Vectastain Elite rat ABC peroxidase kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions and were visualized with diaminobenzidine/H2O2. Total immunostained cells were counted in five ×400 high-power fields (HPFs) in the irradiated skin and expressed as mean ± SEM. Mast cells were identified by staining with low pH toluidine blue and counted in five ×400 HPFs. Significant intergroup differences were determined by applying the two-tailed assuming unequal variance t-test. Immunostaining for keratinocyte-derived cytokine (KC) and macrophage inflammatory protein-2 (MIP-2) were performed using a similar protocol with anti-mouse KC (Antigenix America, Inc., Huntingtion Station, NY) and anti-murine MIP-2 (Chemicon International, Temecula, CA) both at an IgG concentration of 1 μg/ml.

Am J Pathol. 2008 July; 173(1): 68–76.

Figure 1 Influx of neutrophils and macrophages into flank skin of Smad3 WT and KO mice at various times after irradiation. After irradiation flank skin was processed at long times (up to 160 days) (A, B) or short times (up to 48 hours) (C, D) and neutrophils (A, C) and macrophages (B, D) were identified by immunostaining as described in Materials and Methods. The numbers of stained cells in five ×400 fields were counted for each irradiated flank and results are expressed as mean ± SEM. The numbers of flanks processed at each time point were four per genotype in A and B and eight per genotype in C and D. *P < 0.05 for WT (white bars) and KO (black bars) at the same time point. E: Immunostaining of neutrophils in WT (left) and KO (right) dermis 8 hours after irradiation. Arrows point to clusters of neutrophils stained with rat anti-mouse neutrophil antibody and visualized with brown DAB chromagen. Notice the large numbers of neutrophils surrounded by arrows and located in the bottom right corner of the KO (right) panel. DAB with hematoxylin. Original magnifications, ×400. Am J Pathol. 2008 July; 173(1): 68–76.

Reproductive Biology and Endocrinology 2008, 6:29

Isolation of primary Sertoli cells and their culture Sertoli cells were isolated from FM131 mice as previously described [52] with minor modifications. As mature Sertoli cells can not be efficiently cultured, testes from 17 days post-natal (dpn) males were decapsulated in PBS, cut into small fragments and digested in DMEM:Ham's F12 medium (1:1, Gibco BRL, Eggenstein, Germany) containing 2% foetal bovine serum (FBS) (ICN Biomedical, Costa Mesa, CA, USA), 0.2 mg/ml collagenase-dispase (Roche, Mannheim, Germany) and 0.1 mg/ml DNAse I (Roche) for 30 min at 32°C. The resultant seminiferous tubule fragments were washed with DMEM:F12 followed by two additional digestions under the same conditions, and then washed again with DMEM:F12. This material was repeatedly passed through an 18 1/2 G needle and the disaggregated cells were collected by filtration through a 70 μm Cell Strainer (BD Falcon, Lexington, TN, USA). The cells were incubated with continuous shaking in DMEM:F12 containing 2% FBS, 0.4 mg/ml hyaluronidase I-S (Sigma St Louis, MO, USA) and 0.1 mg/ml DNAse I for 30 min at 32°C. The sample was then centrifuged at 200 g for 10 min. The Sertoli cells obtained were resuspended in DMEM:F12 with 10% FBS and allowed to settle (20 min at 32°C). The settled cells were cultured at 32°C in a 5% CO2 atmosphere for three days in DMEM:F12 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1× insulin-transferrin-sodium selenite media supplement (ITSS) (Sigma). The germ cells that had residually attached to the Sertoli cells were removed by hypotonic treatment with 20 mM Tris-HCl, pH 7.4 at 20°C for 3 min, and were cultured in supplemented DMEM:F12 medium. To discern the presence of potential contaminant cells, analysis of transferrin (Trf) expression, as a Sertoli cell marker, was carried out by RT-PCR. As we previously reported to detect other potential contaminant cells RT-PCR analysis was performed for the expression of Hsd17 (17beta-hydroxysteroid dehydrogenase) as a Leydig cell marker and S16 (ribosomal protein) were also assessed as negative and positive controls respectively [53].

The Journal of Neuroscience, January 1, 1998, 18(1):205-213

Figure 5. Histological analysis after 24hr of MCA occlusion. A, Photomicrograph showing the histological changes after 24hr of MCA occlusion in wild-type (Wt) and knock-out mutant mice (Sod2 /+). The infarct area was localized in the caudoputamen and MCA territory cortex in both mice groups. However, cortical infarction extended to the boundary zone of the anterior cerebral artery territory, and brain swelling was extremely severe in the knock-out mutant mice. Also shown are infarct volume (B) and hemisphere enlargement (C) in wild-type and knock-out mutant mice at 24hr ischemia. Values are mean±SE; **p<0.01and ***p<0.001,Student's t test. Cerebral infarction and hemisphere enlargement were significantly more severe in knock-out than in wild-type mice. Scale bar, 1 mm.

J. Clin. Invest. 118(1): 29-39 (2007)

Histological examination. Whole embryos, yolk sac and placenta, were fixed in 4% phosphate-buffered paraformaldehyde (pH 7.2), embedded in paraffin, and cut into 4-μm sections for histological examination. Some yolk sacs were used for immunohistochemical staining with anti-mouse CD31 antibody (BD Biosciences — Pharmingen) to visualize blood vessels. Samples were stained with a Histofine MOUSESTAIN KIT (Nichirei Biosciences) and DAB chromogen and counterstained with methyl green. Apoptosis was visualized in green fluorescence using the TUNEL method with an Apoptosis In Situ Detection Kit (Chemicon) and nuclei were stained with Hoechst 33342. To evaluate the aortic wall structure, immunohistochemical staining was performed using anti-mouse type IV collagen antibody (Collaborative Research), phalloidin, and DAPI (Roche Diagnostics). Confocal microscopic observation was then carried out using a Leica TCS-SP2 laser scanning microscope.

J. Clin. Invest. 118(1): 51-63 (2007)

Tumor escape in a Wnt1-dependent mouse breast cancer model is enabled by p19Arf/p53 pathway lesions but not p16Ink4a loss J. Clin. Invest. Michael T. Debies, et al. 118:51 doi:10.1172/JCI33320 [Go to this article.] Figure 1Mechanisms of mammary tumor relapse in MTB/TWNT mice. (A) Mammary expression of putative Wnt pathway target genes. Northern hybridization analyses are shown. Mammary gland RNA came from 5-week-old virgin female MTB/TWNT mice that were either Dox naive (Off Dox) or treated with Dox for 96 hours (On Dox). Tumor RNA came from clonally related outgrowths derived from a Dox-dependent MTB/TWNT tumor that was explanted onto the flanks of Dox-treated host mice. Paired flank explants were harvested during ongoing Dox treatment or after timed Dox withdrawal. (B) Tumor gene expression patterns. Northern hybridization analysis was performed on RNA samples from primary and relapsed MTB/TWNT mammary tumors. Three relapsed DITs expressed Wnt1 transgene in an inducer-independent manner (lanes marked T), and 3 expressed aberrant transcripts encoding activated β-catenin variants (β). (C) Molecular genetic analysis of the Ctnnb1 (β-catenin) gene. Segments of transcripts encoding the regulatory domain of β-catenin were amplified from tumor-derived RNA via RT-PCR and subjected to DNA sequencing. The coding region of mouse β-catenin is shown schematically, with arrows indicating the primers used for RT-PCR placed relative to their approximate annealing sites along the open reading frame. The blowup depicts critical aa residues encoded within exon 3; known hot spots for cancer-associated aa substitutions are in b. Asterisks denote the residue affected by the S33Y mutation identified in 2 DITs. The upper chromatograms show detection of only the wild-type β-catenin allele in an antecedent primary tumor but additional detection of the S33Y allele in a descendant recurrent tumor. The lower panels depict RT-PCR–based detection of an aberrantly spliced β-catenin transcript lacking exon 3 within a relapsed tumor (R) and not within the antecedent primary tumor (P). (D) Tumor histology. Photomicrographs of H&E-stained sections derived from representative primary-relapse tumor pairs. The mode of tumor escape identified for each relapse is indicated. Scale bar: 50 μm.

J. Clin. Invest. 118(1): 64-78 (2007)

Histologic analyses. Mammary glands and tumors were harvested at the indicated time points and fixed in 10% neutral buffered formalin (Fisher Scientific) for 24 hours at 4°C. Whole-mount hematoxylin staining of mammary glands and H&E staining of 7-μm mammary gland tissue sections was performed as described previously (65). Immunohistochemical staining for EphA2 and PCNA was performed as described previously (17), and proliferation was quantified by calculating the average percentage of PCNA+ nuclei relative to total nuclei (4 random fields of at least 4 independent mammary and tumor samples per genotype; original magnification, ×20). Apoptosis assays were performed using the Apoptag red in situ apoptosis detection kit per the manufacturer’s protocol (Chemicon International). Apoptosis was calculated as the average percentage TUNEL+ nuclei relative to total nuclei (4 random fields of at least 4 independent mammary and tumor samples per genotype; original magnification, ×20). We detected p-Erk in tissue sections using rabbit monoclonal anti–p-Erk antibody clone 20G11 per the manufacturer’s protocol (Cell Signaling Technology). Colorimetric immunohistochemical staining for vWF was performed by the Vanderbilt University Immunohistochemistry Core Facility, and immunofluorescence staining was performed as described previously (8). Microvascular density was determined by counting the number of vWF+ vessels in 4 random fields per sample of at least 4 independent tumors per genotype (original magnification, ×20). ErbB2 immunohistochemistry was performed using 5 μg/ml rabbit anti-ErbB2 antibody (Neomarkers/Lab Vision Corporation).

J. Clin. Invest. 118(1): 89-99 (2007)

Immunohistochemistry of paraffin-embedded and frozen tissues. Paraffin-embedded tumor tissues were sectioned (5 μm thick), mounted on poly-l-lysine-coated glass slides, and allowed to dry overnight at 23°C. These sections were used for detection of PCNA and apoptosis by the ApopTag TUNEL assay, as previously reported (50, 51).

J. Clin. Invest. 118(1): 124-132 (2007).

Histology. Hearts were fixed in 4% paraformaldehyde in phosphate-buffered saline, embedded in paraffin, and sectioned at 5-μm intervals. H&E staining, Masson’s trichrome staining, and picrosirius red staining were performed using standard procedures.

J. Clin. Invest. 118(1): 124-132 (2007).

Measurements of mean cardiomyocyte area and myocardial fibrosis. Cardiomyocyte size was assessed on H&E-stained sections using ImageJ software (http://rsb.info.nih.gov/ij/) (NIH). About 100–150 randomly chosen cardiomyocytes from each group (n = 2–3) were analyzed to measure cross-sectional cardiomyocyte area. ImageJ software (NIH) was used to measure the amount of myocardial fibrosis on picrosirius red stained sections. About 30–50 randomly chosen frames from each group (n = 2–3) were analyzed.

J. Clin. Invest. 118(1): 205-216 (2007)

Histology. For histological assessment of arthritis, total ankle joints from 15-week-old mice were isolated and fixed for 4 days in 4% formaldehyde, then decalcified in 5% formic acid and embedded in paraffin. Tissue sections of 7 μm were stained using H&E to study the inflammatory cell influx and chondrocyte death or using safranin O to determine proteoglycan depletion and cartilage and bone destruction. Each parameter was scored on a scale from 0 to 3 by 2 independent observers in a blinded manner. At least 4 junctions per tissue section and 4 sections per ankle joint were scored.

Gut. 2004 May; 53(5): 634–640.

Histology The gold standard used to establish the accuracy of endoscopic and endosonographic staging examinations was histological examination of the endoscopically or surgically resected tumour. The histological criteria used for early oesophageal carcinoma were the WHO criteria and the Vienna classification.25,26 Generally, the resected specimens were fixed in 4% formalin, dehydrated in gradual alcohol series, and embedded in paraffin. Serial step sections of 4 μm were cut and stained with haematoxylin and eosin and/or alcian blue, pH 2.5. The histological assessment was carried out by experienced pathologists (MS, MV). After resection, specimens were examined with regard to tumour depth (involvement of the mucosa or submucosa)—the principal point of this study. Other histological assessment criteria were: freedom from tumour at the base and lateral margins of the specimens; degree of differentiation (grade G1, well differentiated; G2, moderately differentiated; G3, poorly differentiated); and in some cases invasion of the lymphatic vessels and veins.

Ann Thorac Surg. 2008 April; 85(4): 1271–1277.

Histologic Examination Biopsy specimens were obtained from cadaveric RAs and discarded portions of clinical RAs to confirm the diagnosis of vascular trauma. To exactly calibrate the OCT images against the corresponding histopathologic sections, the vessel site where the biopsy specimen was obtained was marked externally at the location of the catheter, visualized by gross examination of the infrared light at the catheter tip (13). These image-guided biopsy specimens were embedded and frozen in cutting compound (Tissue-Tek O.C.T., Redding, CA), then sectioned at 5 μm and analyzed for endothelial integrity as described [15].

Ann Thorac Surg. 2008 April; 85(4): 1271–1277.

Ex Vivo Radial Artery Perfusion Analysis To simulate the effects of grafting into the coronary circulation, 10 cadaveric RAs with minor ostial intimal tears were perfused ex vivo after harvest with Hank's solution for 30 minutes using a pulsatile perfusion pump (Masterflex L/S, Cole-Parmer Instrument Co, Vernon Hills, IL), as described [16]. Flow into the RA was initiated at 50 mL/min and titrated to maintain pressure of 80 to 110 mm Hg, as monitored by a pressure transducer catheter (Mikro-Tip, Millar Instruments, Inc, Houston, TX).