Isolation of kidney glomeruli from mice  

Isolation of mice glomeruli
1. Mice were anesthetized by an intraperitoneal injection of Avertin (2,2,2-tribromoethyl and tertiary amyl alcohol; 17 μl/g mice) and perfused with 8 × 10⁷ Dynabeads diluted in 40 ml of phosphate-buffered saline through the heart.
2. The kidneys were removed, minced into 1-mm³ pieces, and digested in collagenase (1 mg/ml collagenase A, 100 U/ml deoxyribonuclease I in HBSS) at 37°C for 15 minutes (for newborn mice) or 30 minutes (for adult mice) with gentle agitation.
3. The collagenase-digested tissue was gently pressed through a 100-μm cell strainer using a flattened pestle and the cell strainer was then washed with 5 ml of HBSS.
4. The filtered cells were passed through a new cell strainer without pressing and the cell strainer washed with 5 ml of HBSS.
5. The cell suspension was then centrifuged at 200 × g for 5 minutes.
6. The supernatant was discarded and the cell pellet was resuspended in 2 ml of HBSS.
7. Finally, glomeruli containing Dynabeads were gathered by a magnetic particle concentrator and washed for at least three times with HBSS.
8. During the procedure, kidney tissues were kept at 4°C except for the collagenase digestion at 37°C.

Morphological Studies
1. Dynabead-perfused kidneys were snap-frozen for cryostat sectioning. Sections were stained with hematoxylin and eosin (H&E) and were examined by light microscopy.
2. Isolated glomeruli were examined by both light and electron microscopy (EM). Specimens for EM were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde.
3. Glomeruli intended for transmission EM were subjected to ferrocyanide-reduced OsO4, dehydration, and plastic infiltration before ultrathin sectioning.
4. For scanning EM glomeruli were osmicated according to the OTOTO protocol and dried using hexamethyldisilazane evaporation.  

Flow chart of the isolation technique of mice glomeruli.

Reference
1. Friedman PL, Ellisman MH: Enhanced visualization of peripheral nerve and sensory receptors in the scanning electron microscope using cryofracture and osmium-thiocarbohydrazide-osmium impregnation. J Neurocytol 1981, 10:111-131.
2. Braet F, Dez Anger R, Wisse E: Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells. J Microsc 1997, 186:84-87.