{"id":565,"date":"2013-09-04T17:48:53","date_gmt":"2013-09-04T15:06:07","guid":{"rendered":"http:\/\/semmelweis-egyetem.hu\/immun-patofiziologia\/?page_id=565"},"modified":"2026-01-23T16:49:51","modified_gmt":"2026-01-23T15:49:51","slug":"methods","status":"publish","type":"page","link":"https:\/\/semmelweis.hu\/immun-patofiziologia\/en\/methods\/","title":{"rendered":"Methods"},"content":{"rendered":"

Get to know our methods:<\/h2>\n
\n
In vivo<\/a><\/span><\/p>\n
\n

Mouse models used by us:<\/strong><\/span>
\n\u2022 breast cancer isograft (4T1 cell\/Balb\/C mouse)
\n\u2022 ischemia-reperfusion induced renal fibrosis (NMRI outbread mouse)<\/p>\n

Surgical and chemical models:<\/strong><\/span>
\nEndotoxin-ischemia cross-tolerance mouse model<\/strong><\/em>: Resistance triggered with increasing doses of endotoxin.
\nEndotoxin shock mouse model<\/strong><\/em>: Introduction of the lipopolysaccharides of gram negative bacteria in order to trigger endotoxin shock.
\nDiabetes model<\/strong><\/em>: Type one diabetes mellitus model: Streptozotocin induced inzulin-deficiency model in mice and rats.
\nOrgan transplant<\/strong><\/em>: Ortotropic kidney transplant in rats.
\nSubtotal nephrectomy<\/strong><\/em>: Chronic kidney fibrosis model: we resect the total right kidney and the 2\/3 of the left kidney, resulting a 5\/6 nephrectomy.
\nIschemia-reperfusion<\/strong><\/em>: Left kidney is isolated from the circulation by compressing the renal artery and vein. The right kidney is resected as control. (mice and rats)
\nRenal vein and portal injections<\/strong><\/em>: High volume venous infusion of the given organ for renal tubular and hepatocyte transfection.
\nHydrodynamic treatment<\/strong><\/em>: Rapid (less than 10 ms) venous injection of a high liquid volumen (at least 50% of the circulating volumen) in order to transform abdominal organs with high blood flow (kidneys, liver).<\/p>\n

Functional measurements:<\/strong><\/span>
\nDiuretic cage<\/strong><\/em>: 24 hour collection of urine for proteinuria and albuminuria tests.<\/p>\n

Glomerular damage tests:<\/strong><\/span>
\nAlbuminuria<\/strong><\/em>: Albumine ELISA essay.
\nTotal urine protein<\/strong><\/em>: Total urine protein test on collected urine samples. Mass separation with SDS-PAGE and densitometry.
\nInulin, PAH, mannitol clearance<\/strong><\/em>: Glomerular filtration rate and renal plasma flow fuctional tests.
\nCREA\/UREA<\/strong><\/em>: Blood and urine creatine and urea tests with Reflotron system (Roche).<\/p>\n

Tubular damage tests:<\/strong><\/span>
\nNGAL<\/strong><\/em>: Urine NGAL (neutrophil gelatinase associated lipocalin) evaluation with ELISA
\ngamma-GT\/CREA: Urine creatinine and gamma-GT with Reflotron system (Roche).
\nMicroproteinuria<\/strong><\/em>: SDS-Page and densitometry in order to evaluate low molecular weight proteins in the urine.
\nFunctional tests of the vessels:-
\nBlood pressure<\/strong><\/em>: Telemetry and IITC (piezoelectric) tail blood pressure measurements.<\/p>\n

Functional monitoring of the glucose system:<\/strong><\/span>
\nSerum glucose<\/strong><\/em>: Serum glucose measurement with Reflotron system (Roche).
\nOGTT<\/strong><\/em>: Oral Glucose Tolerance Test.
\nFructosamine<\/strong><\/em>: Serum glycated protein measurement.
\nLiver function<\/strong><\/em>: Serum GGT\/ALAT (GOT)\/ASAT (GPT)\/ALP\/TG\/CHOL with Reflotron system (Roche).<\/p>\n<\/div>\n<\/div>\n<\/div>\n

\n
Ex vivo<\/a><\/span><\/p>\n
\n

Superoxide-anion tests<\/strong><\/em>: Dihydroetidium staining of tissue samples and 3D evaluation of the layers. Detection of superoxide production with confocal microscopy.
\nAorta function measurement<\/strong><\/em>: Endothel function test on mouse aorta, measurements of contraction and relaxation triggered by farmacons in organ chamber.
\nMicrovessel function measurements<\/strong><\/em>: Measurements of reactions to farmacons on isolated resistance vessels in organ chamber.<\/p>\n<\/div>\n<\/div>\n<\/div>\n

\n
In vitro<\/a><\/span><\/p>\n
\n

Molecular biology:<\/strong><\/span>
\nStandard procedures of molecular biology<\/strong><\/em>: PCR, Northern blot, enzymatic digestion, DNA\/RNA isolation from tissue or cells, primer construction.
\nGenotyping<\/strong><\/em>: Genotyping of the bred laboratory animals.
\nRT-PCR<\/strong><\/em>: Quantitative measurement of the mRNA levels in isolated RNA samples in order to evaluate the level of gene expression.<\/p>\n

siRNA:<\/strong><\/span>
\nElectroporation<\/strong><\/em>: Evaluation of the effect of siRNA on gene expression.
\nHydrodinamic treatment<\/strong><\/em>: Venous injection of siRNA in mice in order to examine the effect of siRNA in vivo.<\/p>\n

Complement system:<\/strong><\/span>
\nComplement activation and inhibiton<\/strong><\/em>: Functional and quantitative tests of the serum complement system with ELISA.<\/p>\n

Protein expression:<\/strong><\/span>
\nExamination of the expression level changes of proteins on cell or tissue lysate using Western Blot or ELISA.<\/p>\n

Cell cultures:<\/strong><\/span>
\nPrimer cell culture<\/strong><\/em>: HUVEC (Human Umbilical Vein Endothelial Cells) primer cells isolated from the umbilical chord.
\nFlow cytometry<\/strong><\/em>: Flow cytometry procedures for cell typing using surface markers.
\nIsolation of blood cells: Isolation of red blood cells, granulocytes and lymphocytes from whole blood.
\nLymphocyte activation<\/strong><\/em>: Activation of the isolated lymphocytes with PMA or ionomycin, measurement of the produced cytokines with ELISA.
\nOGD on endothel cells<\/strong><\/em>: Evaluation of the effect of oxygen and glucose withdrawal on the superoxide production in HUVEC cells. (dihydroethidium staining, Life-Death kit confocal microscopy).<\/p>\n

Pathology:<\/strong><\/span>
\nHeart and aortic morphology<\/strong><\/em>: Structural examination of mouse heart muscle cells on half-thin tissue sections using point counting stereology. Digital measurements of the intima thickening in mouse aorta.
\nOrgan perfusion<\/strong><\/em>: Perfusion of the organs in order to acquire samples for histology and molecular biology.
\nStandard histochemical stainings<\/strong><\/em>: Staining of the paraffine embedded samples using PAS, hematoxylin-eosin, Trichrome, Sudan IV, Masson, Oil red O etc.
\nImmunohistology<\/strong><\/em>: C3, fibrotic markers (collagen, fibronectin), and TGF-beta detection on samples using immunohistological methods.<\/p>\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"

Get to know our methods: In vivo<\/a> Mouse models used by us: \u2022 breast cancer isograft (4T1 cell\/Balb\/C mouse) \u2022 ischemia-reperfusion induced renal fibrosis (NMRI outbread mouse) Surgical and chemical models: Endotoxin-ischemia cross-tolerance mouse model: Resistance triggered with increasing doses of endotoxin. Endotoxin shock mouse model: Introduction of the lipopolysaccharides of gram negative bacteria in order to …<\/p>\n","protected":false},"author":162,"featured_media":0,"parent":0,"menu_order":3,"comment_status":"closed","ping_status":"open","template":"","meta":{"_acf_changed":false,"footnotes":""},"categories":[],"tags":[],"class_list":["post-565","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/pages\/565","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/users\/162"}],"replies":[{"embeddable":true,"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/comments?post=565"}],"version-history":[{"count":9,"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/pages\/565\/revisions"}],"predecessor-version":[{"id":2411,"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/pages\/565\/revisions\/2411"}],"wp:attachment":[{"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/media?parent=565"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/categories?post=565"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/semmelweis.hu\/immun-patofiziologia\/wp-json\/wp\/v2\/tags?post=565"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}