Examining how poor diet damages bloodvessels unileipzig
-butyldimethylchlorosilane and incubation for 60 min at 80 °C. Metabolites were measured using a DB5-MS GC column in a 7890 GC system combined with a 5977 MS system .Obese and chow animals were killed and intracardially perfused with 15 ml of PBS and 15 ml of freshly prepared paraformaldehyde, pH 7.4 . Tissues were then processed as described below.pieces and fixed with 4% formaldehyde .
Immunostaining was performed using antibodies for CD62P and CD31 . Sections were deparaffinized and hydrated followed by postfixation in ice-cold acetone for 1 min and washing in PBS for 10 min. Antigen retrieval was performed using antigen-unmasking solution . Sections were incubated for 1 h in blocking solution , followed by the addition of primary antibodies overnight at 4 °C.
Immunostaining was performed on cryosections. Slices were incubated in PBS for 30 min and permeabilized in 0.5% Triton X-100 in PBS for 30 min. Sections were washed three times for 5 min each in PBS and blocked in blocking solution , 10% glycine and 0.2% Triton X-100 ) for 2 h. Primary antibodies were incubated for 48 h at 4 °C ; anti-integrin-β1, 1:50 ) in blocking solution.
Sections were generated using a cryostat and attached to Superfrost slides. Cryosections were fixed with freshly prepared 4% paraformaldehyde, pH 7.4 for 30 min, permeabilized with 0.2% Triton X-100 in PBS for 15 min and blocked in a solution composed of 5% BSA, 10% glycine and 0.2% Triton X-100 in PBS for 1 h. Incubation with anti-CD31 was performed overnight in blocking solution at 4 °C.
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