Colour was developed using the AEC Substrate Program (DAKO) and also the sections have been counterstained with Mayer’s hematoxylin (DAKO). For determination of 6 His-VEGF165 protein expression, the sections were incubated with anti6 His antibody and corresponding fluorescein isothiocyanate-conjugated immunoglobulin (Sigma Chemical Co.). Immunofluorescence signal was evaluated using a Nikon Optiphot epifluorescence microscope (Nikon Inc., Garden City, NY) with an Omega filter fluorescein isothiocyanate/Tex Red.VEGF mRNA Expression by RT-PCRVEGF mRNA levels in ulcerated esophageal SDF-1/CXCL12 Proteins Recombinant Proteins tissue were substantially enhanced versus Integrin alpha-6 Proteins web nonulcerated esophageal tissue 1 day just after ulcer induction. 3 and 7 days immediately after ulcer induction, VEGF mRNA levels in ulcerated esophageal tissue have been increased 240 and 210 , respectively, versus the corresponding nonulcerated esophageal tissue of sham-operated rats (Figure 2).VEGF Protein Expression by Western BlottingWestern blotting with particular anti-VEGF antibody demonstrated the presence of the secreted type of VEGF protein, VEGF165, in nonulcerated rat esophageal tissue (Figure three). A single day right after ulcer induction, VEGF165 protein levels in ulcerated esophageal tissue were not signifi-Assessment of AngiogenesisTo identify microvessels, enhanced polymer one-step staining27 with monoclonal mouse antibody against Fac-1452 Baatar et al AJP October 2002, Vol. 161, No.Figure 1. Western blot detection of HIF-1 and HIF-1 protein expression in ulcerated (UL) esophageal tissue versus nonulcerated esophageal tissue from sham-operated (SO) rats 1, 3, and 7 days after ulcer induction or sham operation. Top: Immunoblotting with anti-HIF-1 antibody detected particular 120-kd bands only in ulcerated, but not in nonulcerated esophageal tissue of sham-operated rats. Immunoblotting with anti-HIF-1 antibody detected particular 95-kd bands in each ulcerated and nonulcerated esophageal tissue of sham-operated rats. Bottom: Quantitative data for HIF-1 protein expression in ulcerated esophageal tissue. Information have been obtained by a computerized video analysis in the Western blots. Values are expressed in intensity units and represent signifies SD. For each column, n six.Figure 2. VEGF mRNA expression in ulcerated (UL) and nonulcerated esophageal tissue from sham-operated (SO) rats detected by RT-PCR. Tissues had been obtained 1, three, and 7 days right after ulcer induction or sham operation. Major: RT-PCR goods obtained with use of specific primers that recognize all four isoforms of VEGF mRNA (196 bp) and primers that recognize rat -actin. Bottom: Quantitative data for VEGF mRNA expression. Information were obtained by computerized analysis of amplified PCR items. Every signal was normalized against the corresponding -actin signal along with the results are expressed as a ratio of VEGF/ -actin. Values are indicates SD. For every column, n 6.cantly unique from those in nonulcerated esophageal tissue. However, three and 7 days soon after ulcer induction, VEGF165 protein levels in ulcerated tissue were elevated 310 and 290 , respectively, versus the corresponding nonulcerated esophageal tissue from sham-operated rats (Figure three). The expression of your bigger nonsecreted form of VEGF protein was affected by esophageal ulceration similarly to that of VEGF165 (data not shown).6 His-VEGF165 Protein ExpressionWe detected 6 His-VEGF165 fusion protein by Western blotting in ulcerated esophageal tissue obtained 7 days,HIF-1 and VEGF Protein Expression by ImmunostainingThere was no positive staining for HIF.
