The Journal of Neuroscience, December 5, 2007, 27(49):13446-13456

Image analysis. Images were acquired using a Leica (Bannockburn, IL) SP2 confocal microscope. All images from a given culture were acquired on the same day with identical acquisition settings. Images were then filtered with a median filter in Photoshop and analyzed using OpenLab software (Improvision, Coventry, UK). The number of GluR1 and GluR2 puncta per length of dendrite was counted using an automation in OpenLab by setting a lower threshold limit for pixel intensity for each channel (fivefold higher than background) and then counting the number of objects meeting the threshold criteria per field of view. The intensity of GluR1 and GluR2 staining within synapses was additionally measured on unprocessed data images using ImageJ software. Images used for presentation in Figures 1 and 4 were contrast enhanced for viewing. Regions of interest (ROIs) were defined as distinct puncta positive for both VGlut1 and VGlut2 and either GluR1 or GluR2. The integrated pixel density of GluR1 or GluR2 staining within the ROI was obtained for 50 ROIs in seven fields of view (350 ROIs total) for each animal. Background intensity values for the same ROI area were obtained for each field of view and were subtracted from the GluR intensity values. Pixel intensity values ranged from 0 to 255. All image analysis was done blind to genotype.

Clin Cancer Res. 2006 June 15; 12(12): 3856–3863

Immunohistochemistry Staining was done for automated analysis of melanoma specimens as previously described (29, 30). Briefly, slides were deparaffinized in xylene and transferred through two changes of 100% ethanol. For antigen retrieval, the slides were boiled in a pressure cooker containing 6.5 mmol/L sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked in a mixture of methanol and 2.5% hydrogen peroxide for 30 minutes at room temperature. To reduce nonspecific background staining, slides were incubated at room temperature for 30 minutes in 0.3% bovine serum albumin/1× TBS. Slides were incubated at 4°C overnight in a humidity tray with the primary antibodies [rabbit polyclonal anti-DR5 immunoglobulin G at 1:350 (Oncogene Research, San Diego, CA) and mouse monoclonal anti-DR4 immunoglobulin G at 1:80 (R&D Systems, Inc., Minneapolis, MN)]. To create a tumor mask, slides were simultaneously incubated overnight with a primary anti-S100 antibody (rabbit anti-human S100 at 1:500 for DR4 and mouse anti-human S100 at 1:200 for DR5). Slides were rinsed thrice in 1× TBS/0.05% Tween 20. For TRAIL-R2, slides were incubated for 1 hour at room temperature with goat anti-rabbit horseradish peroxidase (Envision, DAKO Corp., Carpinteria, CA) to identify the target, and goat anti-mouse immunoglobulin G conjugated to Alexa 546 (Molecular Probes, Inc., Eugene, OR) at a dilution of 1:100 to identify the mask. The same technique was used to assess TRAIL-R1 expression, except that goat anti-mouse horseradish peroxidase and goat anti-rabbit immunoglobulin G conjugated to Alexa 546 were used. The slides were washed again as above and incubated for 10 minutes with Cy5 directly conjugated to tyramide (Perkin-Elmer, Boston, MA) at a dilution of 1:50 for primary antibody identification. The slides were rinsed again and coverslips were mounted with ProLong Gold antifade reagent, which contained 4,6-diamidine-2-phenylindole to identify the nuclei.

apoptosis. 2005 may 10(3): 513–524

Immunocytochemistry Primary cardiac myocytes were grown on 8-well chamber slides coated with rat-tail collagen (Becton Dickenson 354630). Cells were infected with reovirus 24−26 h prior to fixation with 3.7% formaldehyde/ phosphate-buffered saline (PBS) for 15 min at room temperature. Cells were subsequently permeablized and blocked with 5% normal goat serum (Vector S1000) in PBS with 0.1% Tween 20 for 2−4 h at room temperature. Cells were incubated overnight at 4°C with antibodies directed against NF-κB (Santa Cruz, sc-8008) at a 1:30 dilution in blocking solution. After washing in PBS/0.1% Tween 20, cells were incubated with secondary anti-rabbit IgG conjugated to FITC (Vector FI 1200) for 1 h at room temperature before being counterstained with Hoechst 33342 (Molecular Probes H 3570) for 3 min at room temperature. Cells were then washed in PBS/0.1% Tween 20, mounted with vectashield (Vector H1000) and digitally imaged using a Zeiss Axioplan2 epifluorescence microscope.

J. Clin. Invest. 118(1): 195-204 (2007).

Immunohistochemistry and TUNEL staining. For Moma-2 and Bcl-XL staining, frozen sections were fixed with acetone/methanol (vol/vol 1:1) for 30 minutes before being transferred to 1× TBS (8.766 g/l NaCl, 6.055 g/l Tris, pH 7.4) for 10 minutes at room temperature. Sections were then immersed in 0.3% H2O2, rinsed, and transferred into 10% normal rabbit serum (Vector Laboratories), followed by staining with rat anti-mouse Moma-2 antibody, biotinylated second antibody, and HRP-avidin conjugates using the ABC kit from Vector Laboratories. Finally, the sections were developed with DAB (Dako North American), and some of the sections were counterstained with hematoxylin (Thermo Electron Corp.). Bcl-XL staining intensity was quantified by using MetaMorph software in circumscribed areas of atherosclerotic lesions. For TUNEL staining, the sections were stained with the TACS In Situ Kit (R&D Systems) based on the manufacturer’s suggestions. Briefly, sections were immerged in PBS for 15 minutes at room temperature, incubated with protease K for 15 minutes at room temperature, transferred into the TdT labeling buffer, and incubated with the TdT labeling reaction mix. Labeling was stopped by transferring sections to the stop buffer, and the sections were incubated in Strep-Fluor. All reagents were provided in the kit.

J. Clin. Invest. 118(1): 173-182 (2007).

Immunohistochemistry. The indirect streptavidin-biotin-peroxidase method was used, and sections were treated with 0.3 % H2O2 for 5 minutes at room temperature. Sections were incubated in primary anti-rabbit AQP4 (1:200) antibodies for 2 h at room temperature. After the immunohistochemical reaction, sections were stained with an HRP IHC kit (Chemicon).

J. Clin. Invest. 118(1): 161-172 (2007)

Immunohistochemistry. Heavily anesthetized animals were perfused with fresh 4% paraformaldehyde in 0.2 M sodium phosphate, pH 7.4. Tissues were harvested, postfixed overnight in perfusate, and processed for paraffin embedding (70, 77). Paraffin sections (10 μm) were cut, deparaffinized, and incubated in antigen retrieval (Dako) for 5 min at 95°C and then for 20 min at room temperature. The sections were then treated in 3% hydrogen peroxide for 5 min. Nonspecific antibody binding was blocked with 10% normal goat serum. Sections were treated with primary antibody overnight at 4°C in 0.1% goat serum in PBS, rinsed, and subsequently incubated for 1 h with biotinylated HRP-conjugated goat anti-rabbit secondary antibody, followed by Avidin Biotin complex (ABC). Sections were developed with 3′3-diaminobenzidine and counterstained with methyl green. Some sections were treated only with secondary antibody as a control. Imaging was done using a Leica microscope and Open Lab software.

J. Clin. Invest. 118(1): 133-148 (2007).

Measurement of LDH activity and immunocytochemistry of MAP-2. In order to prove the concept of neuroprotection of SN, PCCs were prepared in 24-well plates and pretreated with 1 μg/l SN (Neosystems). After 20 minutes of SN pretreatment, PCCs were subjected to OGD in the hypoxia chamber for 4 hours, and then the culture media were collected for LDH activity assays as described previously (46). For MAP-2 immunostaining, PCC cultures were washed with PBS and fixed with 1% paraformaldehyde. The immunostaining procedure with specific antibody against MAP-2 (1:1,000; Boehringer Mannheim) and the method for quantification of MAP-2+ cell density has been described previously (47).

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): 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).

Am J Pathol. 2008 July; 173(1): 30–41.

Immunohistochemical and Immunofluorescence StainingImmunohistochemical staining of kidney sections was performed by an established protocol.34 In brief, paraffin-embedded sections were stained with anti-CD3 (sc-20047, Santa Cruz Biotechnology), anti-F4/80 (14-4801-82; eBioscience Inc., San Diego, CA), and anti-RANTES (500-P118; PeproTech Inc., Rocky Hill, NJ) antibodies using the M.O.M. immunodetection kit, according to the protocol specified by the manufacturer (Vector Laboratories, Burlingame, CA). Indirect immunofluorescence staining was performed according to the procedures described previously.35 Slides were viewed with an Eclipse E600 microscope equipped with a digital camera (Nikon, Melville, NY). Nonimmune normal rabbit IgG was used to replace the primary antibody as negative control, and no staining occurred. Nuclear staining for p65 NF-κB in HKC-8 cells after various treatments was counted and calculated. CD-3, F4/80, and RANTES staining were semiquantified by a computer-aided morphometric analysis (MetaMorph; Universal Imaging Co., Downingtown, PA). Briefly, a grid containing 117 (13 × 9) sampling points was superimposed onto images of cortical high-power field (×400). The number of grid points overlying positive area (except tubular lumen and glomeruli) was counted and expressed as a percentage of all sampling points. For each kidney, 10 randomly selected, nonoverlapping fields were analyzed in a blinded manner.

Reproductive Biology and Endocrinology 2008, 6:40

TLR3 and TLR4 protein is localised to endometrial cells during the menstrual cycle. TLR3 protein staining in healthy late proliferative (LP) tissue was high in luminal and glandular tissue (A, brown precipitate) and lower in LP endometriotic tissue (B). Late secretory (LS) endometrium showed highly expressed TLR3 in the epithelium (C), but weakly in endometriosis (D). Intense staining of TLR4 proteins was shown in mid proliferative (MP) tissue (E). In late proliferative phase of endometriosis, TLR4 proteins were comparably lower (F). TLR4 protein was high in mid secretory (MS) normal endometrium (G), whereas it was decreased in endometriotic MS tissue (H). During the menstrual phase, both TLR3 (I) and TLR4 (J) were highly expressed. Co-immunostaining for TLR4 (green), CD14 (K, red) and CD163 (L, red) demonstrated that TLR4 proteins were expressed by CD14 positive dendritic cells and monocytes (K, yellow) and by CD163 positive macrophages (L, yellow). Localisation of TLR4 to immune cells is marked by a black arrow (J) and by white arrows (K, L). Allhorn et al. Reproductive Biology and Endocrinology 2008 6:40

Reproductive Biology and Endocrinology 2008, 6:49

Immunohistochemical staining for PBX1, PBX2 and MEIS1/2 in human ovaries. (A, B, C, J) PBX1, (D, E, F, K) PBX2 and (G, H, I, L) MEIS1/2. (A, D, G): primordial follicles. (B, E, H): primary follicles. (C, F, I): secondary follicles. (J, K, L): preovulatory follicles. Figure 2E contains both primordial and primary follicles, and Figure 2I contains both primordial and secondary follicles (scale bars: A,B,D,E,G,H = 9 μm, C,F,I = 40 μm, J,K,L = 350 μm). Ota et al. Reproductive Biology and Endocrinology 2008 6:49 doi:10.1186/1477-7827-6-49

Reproductive Biology and Endocrinology 2008, 6:49

Immunohistochemistry Ishikawa, Hela and K562 cell pellets were solidified by collagen gel, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections of these cells and of the ovaries were deparaffinized, rehydrated, and submitted to antigen retrieval by a steamer for 30 min in 10 mM citric buffer (PBX2) or Target Retrieval Solution (DAKO, Mississauga, ON; PBX1 and MEIS1/2). Endogenous peroxide was diminished with 3% H2O2 for 30 min. Nonspecific binding was blocked with 3% BSA in PBS (PBX2) or Protein Block (DAKO; PBX1 and MEIS1/2) for 30 min, followed by incubation with primary antibodies overnight at 4°C. The dilution of the primary antibodies was the same as that described for the immunofluorescence. Samples without primary antibodies were used as a negative control. There was no or little background without primary antibodies. The sections were then incubated with Biotinylated link universal (DAKO) for 15 min and streptavidin (DAKO) for 25 min at 25°C. Slides were developed in diaminobenzine (DAKO) or in NovaRED substrate (Vector Laboratories, Inc., Burlington, ON) and counterstained with hematoxylin.

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

Figure 3 Egr-1 regulates genes involved in gliosis. A: Egr-1 loss-of-function effects on human astrocytes transfected with siRNAs against Egr-1. RT-PCR analysis shows that knockdown of Egr-1 leads to reduction in the expression levels of genes encoding ECM components of the glial scar. c, control mock-transfected cells; −, Egr-1 knockdown. DNA size markers are shown on the left. Gene name abbreviations are as follows: CSPG2, 3, 4, chondroitin sulfate proteoglycan 2, 3, 4, respectively; Lamα1, laminin α1; Lamα2: laminin α2; Lamβ1: laminin β1. Real-time PCR quantification of the effects of the Egr-1 knockdown on the expression of putative gene targets shows a 60% drop in phosphacan RNA levels in Egr-1 siRNA-treated astrocytes. Adjusted for aldolase, relative phosphacan expression was 12.96 ± 1.21 U in controls versus 5.05 ± 0.89 U after Egr-1 knockdown (n = 5, P < 0.001). B–E: Immunofluorescence analysis after Egr-1 overexpression in astrocytes transfected with the CMV-Egr-1-IRES-EGFP construct. Transfected, EGFP-positive cells (green, marked by arrows), stain more intensely with antibodies recognizing laminin α1 (B, red) and phosphacan (C, red, anti-RPTPβ) than nontransfected neighboring cells. No difference in expression levels of GFAP (D, red) or β-tubulin (E, red) between Egr-1-overexpressing cells (green, marked by arrows) and nontransfected cells. Superimposed images (far right) confirm that transfected cells express higher levels of putative Egr-1 targets, but similar levels of other proteins. F: Western blotting of proteins isolated from astrocytes transfected with siRNAs against Egr-1 (Egr-1 siRNA), control siRNA (scrambled siRNA), or mock-transfected cells (c). siRNAs against Egr-1 diminish effectively Egr-1 protein levels and lead to down-regulation of phosphacan (Pcan, detected with the KAF13 antibody). β-Tubulin (β-Tub) levels remain unaffected serving as control. Quantification of blot images shows that Egr-1 protein levels are reduced 3.0-fold versus control (mock-transfected cells; SD, 0.4) and 2.8-fold (SD, 0.2) versus scrambled siRNA-transfected cells; phosphacan protein levels are down-regulated 2.18-fold versus control (mock; SD 0.1) and 2.23 times (SD 0.1) versus scrambled siRNA. G: Western blotting of proteins isolated from astrocytes transfected with the CMV-Egr-1-IRES-EGFP (Egr-1 cDNA) construct or the empty vector (c). Egr-1 protein levels increase 2.08-fold (SD, 0.4) leading to 1.42-fold up-regulation of phosphacan (Pcan; SD, 0.14). β-Tubulin (β-Tub) serves as control. Am J Pathol. 2008 July; 173(1): 77–92.

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

Figure 2 Egr-1 expression in adult mouse brain after cerebral ischemia. A: Cerebral ischemia was induced by permanent occlusion of the MCA. The infarcted tissue appears white in the TTC-stained brain slice (inset). Immunohistochemistry using anti-Egr-1 antibody 4 days after MCAO detects Egr-1 expression in cells within the infarct area (arrows) and around the infarct border zone (star). B–D: Immunofluorescence analysis of brain tissue sections stained with anti-Egr-1 and GFAP antibodies 4 days after MCAO. Egr-1-expressing cells in the border zone (B, green color) stain positive for GFAP (C, red color). D: Double-labeled astrocytic cells appear yellow/orange in the superimposed images. E–G: High Egr-1 expression persists in cells around the injury site 6 weeks after infarction. Low-magnification images reveal strong Egr-1 expression (green) in cells accumulating around the infarct region (E) compared with the corresponding area of the contralateral hemisphere (F), or the ipsilateral area of sham-operated animals (G). H–J: Confocal microscopy images of brain tissue sections stained with anti-Egr-1 and GFAP antibodies 6 weeks after MCAO. Egr-1-positive cells (H, green) around and within the glial scar stain positive for GFAP (I, red). Double-labeled astrocytes appear yellow/orange (J). K and L: Confocal microscopy images of brain tissue sections stained with anti-Egr-1 antibody. K: The number of Egr-1-positive cells (in green) around the ventricles of infarcted hemispheres increases after MCAO (L, arrows) compared with contralateral controls. M: Western blotting detects higher levels of Egr-1 protein in scar tissue isolated from infarcted (left, l) hemisphere as compared with corresponding noninfarcted area of the contralateral side (right, r). β-Actin protein levels serve as control. Sham-operated animals have comparable Egr-1 amounts on both brain sides. Quantitative image analysis shows that Egr-1 protein levels are 2.3-fold higher in the peri-infarct region compared with control tissue (SD, 0.3), whereas sham-operated animals show equivalent amounts of Egr-1 protein in the corresponding areas of both hemispheres (0.98-fold difference; SD, 0.12). i: infarct; dotted lines demarcate infarct and peri-infarct areas. Scale bars: 100 μm (A, E–G, K, L); 25 μm (B–D, H–J). Am J Pathol. 2008 July; 173(1): 77–92.