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

Figure 1 HGF inhibits renal infiltration of inflammatory CD3-positive T cells. A朎: Immunohistochemical staining revealed an increased infiltration of CD3-positive T cells in the obstructed kidney at 7 (B) and 14 (D) days, respectively, after UUO, compared with sham control (A). Delivery of HGF gene effectively inhibited the infiltration of T cells in the obstructed kidneys at 7 (C) and 14 (E) days after UUO, respectively, as indicated. F: Graphic presentations of quantitative data are given. Data were presented as mean ?SEM of five animals per group (n = 5). *P < 0.01 versus sham control. ?/SUP>P < 0.01 versus pcDNA3. Scale bar = 20 靘. Am J Pathol. 2008 July; 173(1): 30?1.

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

Figure 2 HGF inhibits renal infiltration of inflammatory F4/80-positive macrophages. A朎: Immunohistochemical staining revealed an increased infiltration of F4/80-positive macrophages in the obstructed kidney at 7 (B) and 14 (D) days, respectively, after UUO, compared with sham control (A). Delivery of HGF gene effectively inhibited the infiltration of macrophages in the obstructed kidneys at 7 (C) and 14 (E) days after UUO, respectively, as indicated. F: Graphic presentation of quantitative data are given. Data were presented as mean ?SEM of five animals per group (n = 5). *P < 0.01 versus sham control. ?/SUP>P < 0.01 versus pcDNA3. Scale bar = 20 靘.

Am J Pathol. 2008 July; 173(1): 42–56.

Immunohistochemical Analysis. The cellular distribution of FasL protein in lung tissues was studied by the streptavidin-biotin immunoperoxidase method using a polyclonal rabbit anti-FasL antibody (Chemicon/Millipore, Billerica, MA).10,11 Immunoreactivity was detected with 3,3′-diaminobenzidine tetrachloride. Specificity controls consisted of omission of primary antibody.

Am J Pathol. 2008 July; 173(1): 42–56.

Figure 3 Immunohistochemical analysis of FasL protein expression. A: Representative FasL immunostaining of lungs of Dox-treated CCSP+/FasL+ mice at E19 showing diffuse and intense FasL immunoreactivity, localized to alveolar epithelial cells morphologically consistent with type II cells, intra-alveolar cellular debris (arrows), and bronchial epithelial cells. B: Lungs of single-transgenic CCSP+/FasL− littermates showing modest FasL immunolabeling in bronchial epithelial cells and scattered alveolar epithelial type II cells (arrows). C: Negative control (omission of primary antibody). Anti-FasL immunostaining by ABC method, hematoxylin counterstain. Original magnifications, ×400.

Am J Pathol. 2008 July; 173(1): 42–56.

Figure 8 Gross and microscopic appearance of lungs of Dox-treated transgenic mice at P21. A and B: Macroscopic appearance of formalin-inflated lungs of Dox-treated CCSP+/FasL+ and CCSP+/FasL− mice at P21. Lungs of double-transgenic pups appeared large and pale with obtunded edges. C and D: Representative photomicrographs of lungs of Dox-treated CCSP+/FasL+ and CCSP+/FasL− mice at P21. C: CCSP+/FasL+ lungs at P21 showing large-sized simple alveolar spaces separated by thin alveolar septa. Small macrophage collections are present. D: Lungs of CCSP+/FasL− littermate of C showing a complex network of small-sized alveoli (advanced alveolar stage). H&E staining. Original magnifications, ×400. Am J Pathol. 2008 July; 173(1): 42–56.

Am J Pathol. 2008 July; 173(1): 57–67.

Figure 2 IL-1RI-null animals had significantly decreased macrophage infiltration after 24 hours of reperfusion. Immunohistochemical staining with Mac2 identified macrophages infiltrating infarcted WT hearts after 24 hours (A), 72 hours (B), and 7 days (C) of reperfusion. D: IL-1RI-null mice had decreased macrophage infiltration after 24 hours of reperfusion. However, macrophage density in IL-1RI−/− infarcts after 72 hours (E) and 7 days (F) of reperfusion was comparable with WT animals. G: Quantitative analysis of macrophage density in the infarcted heart (*P < 0.05 versus corresponding WT mice). Am J Pathol. 2008 July; 173(1): 57–67.

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.

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

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): 133-148 (2007).

TUNEL histochemistry. To detect cellular apoptosis, a TUNEL staining Kit (DeadEnd Fluorimetric TUNEL system; Promega) was used for the TUNEL assay. Twenty-four hours after ischemia, rat brains were fixed by transcardial perfusion with saline and immersed in 4% paraformaldehyde. After brains had been frozen on dry ice, a series of adjacent 10-μm-thick sections were cut in the coronal plane with a cryostat. The staining and semiquantitating procedure was performed as previously described (61).

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.