Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Epimorphin regulates the intestinal stem cell niche via effects on the stromal microenvironment

doi: 10.1152/ajpgi.00224.2017

Figure Lengend Snippet: Increased stem cell marker gene expression and goblet and Paneth cell populations in Epim−/− vs. wild-type (WT) enteroids. A: Lgr5, Axin2, Wnt3a, and Olfm4 mRNA expression was quantified by qRT-PCR analysis of RNA isolated from WT (gray) and Epim−/− (black) enteroids at 6 days postplating (crypts isolated from n = 4 WT and 4 Epim−/− mice; *P < 0.001 for Lgr5, *P = 0.05 for Axin2, *P = 0.02 for Wnt3a and Olfm4). B: enteroids were fixed, sectioned, stained with hematoxylin and eosin, and analyzed for percentage of goblet and Paneth cells (n = 58 WT and 54 Epim−/− enteroids; *P = 0.01 for goblet cells, *P < 0.001 for Paneth cells). Percentage of enterocytes was determined by hematoxylin and eosin and alkaline phosphatase staining, *P = 0.05.

Article Snippet: Total number of cells, number of goblet cells, Paneth cells, and enterocytes (detected by hematoxylin and eosin and alkaline phosphatase; Vectastain ABC-AP kit, Vector Laboratories, Burlingame, CA) were counted for each enteroid.

Techniques: Marker, Expressing, Quantitative RT-PCR, Isolation, Staining

Journal: Journal of Ovarian Research

Article Title: Proteomic identification of fucosylated haptoglobin alpha isoforms in ascitic fluids and its localization in ovarian carcinoma tissues from Mexican patients

doi: 10.1186/1757-2215-7-27

Figure Lengend Snippet: Fucosylation profile of haptoglobin. A) . Haptoglobin standard (ST), an ascitic fluid sample non-related with cancer (NR), and 40 ascitic fluid samples from ovarian cancer patients were analyzed for fucosylation of haptoglobin alpha. Protein samples (50 μg) from ascitic fluid free of abundant proteins were processed through a 12.5% SDS-PAGE and transferred to a NCP to incubate with biotinylated- Aleuria aurantia lectin (1:1000), specific for fucose residues; variable levels of fucosylation were detected by a colorimetric system using Alkaline Phosphatase-conjugated Streptavidin (1:2000) (lower left panel) or with Horseradish Peroxidase-conjugated Streptavidin (1:1000) (lower right panel). As loading control, the same samples were incubated with an anti-Hp antibody that recognizes the β, α1 and α2 isoforms of Hp to detect total Hp, and a secondary antibody conjugated with Horseradish Peroxidase (1:1000) (upper right panel) or Alkaline Phosphatase-conjugated antibody (1:1000) (upper left panel). B) . Graph showing densitometric values of samples’ fucosylation in arbitrary units, comparing clinical stages IIIC and IV vs. a non-cancer related sample. C) . Fucosylation levels (low, medium or high) of samples, according to clinical stages.

Article Snippet: Following this, the membrane containing the samples from the 15% SDS-PAGE was incubated with alkaline phosphatase-Streptavidin (Vector Labs, Cat. No. SA-5100), and with the substrate for alkaline phosphatase, BCIP/NBT (Cat. No. SK-5400).

Techniques: SDS Page, Incubation

Journal: Journal of Ovarian Research

Article Title: Proteomic identification of fucosylated haptoglobin alpha isoforms in ascitic fluids and its localization in ovarian carcinoma tissues from Mexican patients

doi: 10.1186/1757-2215-7-27

Figure Lengend Snippet: Immunolocalization of haptoglobin and its fucosylation state in ovarian carcinoma. A) . Detection of total (Hp T ) and fucosylated Hp (Hp F ) in a Hp commercial standard (ST) and a tissue extract from an ovarian tumor developed in Nu/Nu mice (T) by Western blot and lectin binding assays (as described in figure ). B, C, E) . Immunolocalization of Hp, fucosylated Hp and fucosylation. Tissue nuclei were stained with DAPI (blue) to help define the tissues’ structures; Hp was stained with a primary anti-Hp antibody and was developed with a secondary TRITC coupled antibody (red); fucosylation was detected by biotinylated- Aleuria aurantia lectin followed by FITC coupled streptavidin (green). All reagents were used at a 1:100 dilution and the scale bar = 100 μm. B) .Histo-immunofluorescence of stromal and epithelial (a) and germinal (b) areas of cancer-free ovarian tissue slices. The analysis of tile scan shows a tridimensional image generated by 25 fields’ caption (left panels); from the tile scan, an area was selected (yellow square) and optical zooms were done twice (10X and 40X). Epithelium (E), stroma (S), and follicles (F). C) . Tumor tissue sections from four different patients. Fucosylation of Hp was analyzed in broad zones (5x5) of tissues as a tile-scan analysis. A representative region (yellow box) of each tissue was selected to detect fucosylated Hp in optical zooms. D) . Graph showing a semi-quantitative analysis of nuclei, Hp and fucosylation content, considering an average area. This analysis was performed with the Software Zen 2011 (blue edition, Carl Zeiss) determining the mean fluorescence intensity for each molecule of interest given in arbitrary units. E) . Immunolocalization of fucosylated Hp in individual cells in an optical zoom from a selected region of a tumor tissue section; the crop section analyzed (yellow box) clearly shows the cytoplasmic distribution of fucosylated Hp with approximately 80% of co-localization found in each selected cell.

Article Snippet: Following this, the membrane containing the samples from the 15% SDS-PAGE was incubated with alkaline phosphatase-Streptavidin (Vector Labs, Cat. No. SA-5100), and with the substrate for alkaline phosphatase, BCIP/NBT (Cat. No. SK-5400).

Techniques: Western Blot, Binding Assay, Staining, Immunofluorescence, Generated, Software, Fluorescence

Journal: Human Molecular Genetics

Article Title: Disrupted mitochondrial function in the Opa3 L122P mouse model for Costeff Syndrome impairs skeletal integrity

doi: 10.1093/hmg/ddw107

Figure Lengend Snippet: Immunolocalisation of Opa3 in mouse tibiae. Immunohistochemical visualisation of Opa3 expression in 6-month-old WT tibiae with (A,C,E,G,I,K,M,O) and without (B,D,F,H,J,L,N) application of the primary rabbit anti-mouse Opa3 antibody, using the ImmPRESS peroxidase secondary antibody in conjunction with BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue terazolium) peroxidase substrate to produce indigo labelling, all sections being counterstained with haematoxylin and eosin. The immunoreactivity observed under low power ( A ) was located in osteoblasts on the endosteal (black arrowheads) and trabecular (grey arrowheads) (C), surfaces and on the surface of the cortical periostium (white arrowheads; E) and synovial cartilage (light grey arrowheads; G). In addition, Opa3 immunoreactivity was observed in cortical osteocytes (black arrows; E), synovial chondrocytes (dark grey arrows; G), germinal (g), proliferative (p) and hypertrophic (h) growth plate chondrocytes (I,K), mesenchymal stem cell rosettes (white arrows) in the mid-diaphyseal marrow (M) and adipocytes in the proximal (*; I,K) and distal (v;O) marrow (Scale bars: 300 µm (A,B), 50µm (C,D,I,J,M,N,O) and 20 µm (E,F,G,H,K,L).

Article Snippet: A rabbit anti-human/mouse/rat Opa3 primary antibody (optimal dilution 1:100; Cat # 15638-1-AP; ProteinTech, Chicago, IL, USA) in combination with the ImmPRESS peroxidase secondary antibody and the BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue terazolium) peroxidase substrate (Cat #s MP-7401 & SK-5400; Vector Laboratories Inc., Burlingame, CA, USA) was used to visualise Opa3 expression, the tissue being counterstained with haematoxylin and eosin.

Techniques: Immunohistochemical staining, Expressing