Lmd6000 laser microdissection microscope

Kidneys were extracted after euthanasia. Sections from each kidney were snap frozen in OCT compound (Tissue-Tek, Cat. #4583) on dry ice and kept at −80° C until use. They were subsequently cut using a cryotome at 10 μm sections and mounted on Leica PPS membrane slides (Leica, Cat. #11505268). The protocol for staining S3 proximal tubules, described previously in detail (17 ), identifies these tubules by the intense brush border stain in the outer medulla. Following rapid dehydration/re-hydration steps the samples were stained with FITC-phalloidin (1:20) + DAPI (Molecular Probes, 1:300), washed with PBS and the membranes were allowed to air dry. Following staining, sections were immediately taken to a Leica LMD6000 Laser micro-dissection microscope. Dissection was performed at 40x magnification under fluorescence. There were 200–250 segments dissected in each 90-minute session (average dissected area varies on type of tubules, for S3 segments this ranges from 350,000 – 500,000 μm²). RNA extraction was performed using Arcturus PicoPure RNA isolation kit (Applied Biosystems).

For laser capture microdissection (LCM), bladder sections were prepared using an LCM staining kit (Ambion) and a Leica LMD6000 Laser Microdissection Microscope. After LCM, total RNA was prepared using RNAqueous-Micro RNA isolation kit (Ambion). Quantitative RT-PCR was performed using iScript one step RT-PCR kit with SYBR Green and the Bio-Rad iCycler (BioRad). All values normalized to the HPRT internal control.