Expression, qPCR, based on the Taqman fluorescence method (ABI Prism 7000 sequence detection system; Life Technologies), was used. Taqman probes and primers for GAPDH (Mm99999915_g1), collagen I 1 (Mm00801666_g1), TGF1 (Mm01178820_m1), CCN2 (Mm01192931_g1) CCN3 (Mm00456855_m1), Havcr1 (Mm00506686_m1), Lcn2 (Mm01324470_m1), TNF (Mm00443258_m1) and IL-6 (Mm00446190_m1) were purchased from Life Technologies (Gent, Belgium). Each gene was analysed in triplicate and the expression was normalized to the reference gene GAPDH. Calculations were made conform the comparative CT-method. Histology. Renal morphology was evaluated on ischemic kidney AZD0156 site tissue fixated in NBF (Neutral Buffered Formalin), stained with Masson’s trichrome after post-fixation in Bouin’sPLOS ONE | DOI:10.1371/journal.pone.0152153 March 23,5 /An Ischemic Mouse Model for AKI to CKDfixative. Masson’s trichrome stain is the standard for visualizing fibrosis in tissue, provides a useful sense of tissue morphology, and allows evaluation of localization and severity of deposition of extracellular matrix. For collagen I immunostaining, paraffin embedded 4 m thick sections of ischemic kidney tissue were blocked with normal goat serum and PG-1016548 web incubated overnight with the primary antibody, polyclonal rabbit anti-mouse collagen I antibody (dilution 1/3500,Catalogue number T40777R, Lot number 20I25000, Biodesign International, Saco, Maine). After washing, sections were incubated with a biotinylated secondary antibody, goat anti-rabbit IgG antibody (dilution 1/200, PK-4001, Vector Laboratories, Burlingame, California) and subsequently incubated with avidin and biotinylated horseradish peroxidase (AB-complex, Vector Laboratories, Burlingame, California). A dark brown colour was developed with diaminobenzidine in the presence of 3 H2O2. Sections were counterstained with methyl green to visualize nuclei. Collagen I immunostaining was quantified using the Axiovision image analysis software (Carl Zeiss, Jena, Germany) and quantification was performed blinded. The area stain represents the ratio of the summed absolute areas of staining versus the total tissue. Statistics. All statistical analysis was performed with SPSS Statistics 22 (IBM, Brussel, Belgium). Data are presented as mean ?standard deviation, or as individual values. Comparisons between groups are assessed using a Kruskal-Wallis test, followed by a two-tailed Mann-Whitney U test. Values of p<0.05 are considered significant.Results of experiments for the evaluation of unilateral fpsyg.2017.00209 IRI without nephrectomy as initiator of CKD Technical survey and considerations on the ischemia-reperfusion procedureAnaesthesia. General anaesthesia in laboratory animals involves loss of consciousness, loss of sensation (analgesia) and muscle relaxation [46]. An ideal anaesthetic agent is easy to administer, produces a fast and adequate immobilization, has limited side effects, and is reversible and safe for animals and operators. Unfortunately such an anaesthetic is not available, and the best drug selection is highly variable according to different experimental circumstances [47] e.g. interference with pathology. Inhalation anaesthesia is usually preferred to SART.S23503 injection anaesthetics. Induction and recovery of inhalation anaesthesia are rapid, safer (as it causes less cardiovascular depression) and allows accurate control over the depth of anaesthesia [45]. However, compared to injection anaesthesia, inhalation anaesthetics are counter-indicated for use duri.Expression, qPCR, based on the Taqman fluorescence method (ABI Prism 7000 sequence detection system; Life Technologies), was used. Taqman probes and primers for GAPDH (Mm99999915_g1), collagen I 1 (Mm00801666_g1), TGF1 (Mm01178820_m1), CCN2 (Mm01192931_g1) CCN3 (Mm00456855_m1), Havcr1 (Mm00506686_m1), Lcn2 (Mm01324470_m1), TNF (Mm00443258_m1) and IL-6 (Mm00446190_m1) were purchased from Life Technologies (Gent, Belgium). Each gene was analysed in triplicate and the expression was normalized to the reference gene GAPDH. Calculations were made conform the comparative CT-method. Histology. Renal morphology was evaluated on ischemic kidney tissue fixated in NBF (Neutral Buffered Formalin), stained with Masson’s trichrome after post-fixation in Bouin’sPLOS ONE | DOI:10.1371/journal.pone.0152153 March 23,5 /An Ischemic Mouse Model for AKI to CKDfixative. Masson’s trichrome stain is the standard for visualizing fibrosis in tissue, provides a useful sense of tissue morphology, and allows evaluation of localization and severity of deposition of extracellular matrix. For collagen I immunostaining, paraffin embedded 4 m thick sections of ischemic kidney tissue were blocked with normal goat serum and incubated overnight with the primary antibody, polyclonal rabbit anti-mouse collagen I antibody (dilution 1/3500,Catalogue number T40777R, Lot number 20I25000, Biodesign International, Saco, Maine). After washing, sections were incubated with a biotinylated secondary antibody, goat anti-rabbit IgG antibody (dilution 1/200, PK-4001, Vector Laboratories, Burlingame, California) and subsequently incubated with avidin and biotinylated horseradish peroxidase (AB-complex, Vector Laboratories, Burlingame, California). A dark brown colour was developed with diaminobenzidine in the presence of 3 H2O2. Sections were counterstained with methyl green to visualize nuclei. Collagen I immunostaining was quantified using the Axiovision image analysis software (Carl Zeiss, Jena, Germany) and quantification was performed blinded. The area stain represents the ratio of the summed absolute areas of staining versus the total tissue. Statistics. All statistical analysis was performed with SPSS Statistics 22 (IBM, Brussel, Belgium). Data are presented as mean ?standard deviation, or as individual values. Comparisons between groups are assessed using a Kruskal-Wallis test, followed by a two-tailed Mann-Whitney U test. Values of p<0.05 are considered significant.Results of experiments for the evaluation of unilateral fpsyg.2017.00209 IRI without nephrectomy as initiator of CKD Technical survey and considerations on the ischemia-reperfusion procedureAnaesthesia. General anaesthesia in laboratory animals involves loss of consciousness, loss of sensation (analgesia) and muscle relaxation [46]. An ideal anaesthetic agent is easy to administer, produces a fast and adequate immobilization, has limited side effects, and is reversible and safe for animals and operators. Unfortunately such an anaesthetic is not available, and the best drug selection is highly variable according to different experimental circumstances [47] e.g. interference with pathology. Inhalation anaesthesia is usually preferred to SART.S23503 injection anaesthetics. Induction and recovery of inhalation anaesthesia are rapid, safer (as it causes less cardiovascular depression) and allows accurate control over the depth of anaesthesia [45]. However, compared to injection anaesthesia, inhalation anaesthetics are counter-indicated for use duri.