Computational protocol: A Renal Olfactory Receptor Aids in Kidney Glucose Handling

Similar protocols

Protocol publication

[…] WT and KO mouse kidneys were perfusion fixed in 4% (vol/vol) PLP, and 8 μM cryosections were prepared and stained for Sglt1 or Sglt2. Kidney sections were subjected to antigen retrieval in a 10 mM citrate buffer at 70 °C for 20 mins, followed by Tx-100 permeabilization (0.5% for 15 min and 2% for 30 min). After blocking in 1% BSA, sections were incubated with the polyclonal Sglt1 or Sglt2 antibody (1:500) overnight at 4 °C. The sections were then washed with PBS and Sglt staining was detected with alexa 594 secondary antibodies. Proximal tubules were labeled with LTL (Vector Labs; Burlingame, CA) and post-fixed with 4% paraformaldehyde. Confocal images were taken with a Zeiss AxioObserver LSM700 at 40X and the Z stacks were compressed with Zen software. To quantify the amount of luminal stain, random cortical fields from each kidney section were visualized and, for each LTL+/Sglt+ tubule within the field of view, the average pixel intensity was determined for (a) the luminal region and (b) whole tubule. Analysis was done using ImageJ (National Institutes of Health; Bethesda, MD). The ratio of the luminal/total Sglt stain was then determined for each tubule. The ratio from all tubules analyzed per kidney (n = 10–42 tubules) were averaged. While representative images from 2 kidneys are shown in , , the quantification represents staining from 4–6 different animals/genotype. [...] The student t-test was performed on all in vivo assays to determine differences between WT and KO mice (P < 0.05 considered significant). Olfr1393 activation by ligands was determined by a significant difference in the Firefly: Renilla ratio as compared to the non-treated control (student t-test; p < 0.05). EC50 values were calculated using SigmaPlot. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Kidney Diseases
Chemicals Glucose