Microsystems microtome

For electron microscopy the selected part of the mouse kidney was transferred from 4% formaldehyde into a cacodylate buffer with sucrose for 10 min at 80°C. Afterwards, osmium tetroxyde was applied for 2 h. The specimen was washed in cacodylate buffer plus sucrose twice for 5 min. Subsequently, the sample was contrasted with uranyl acetate for 1 h. The specimen was put into ethanol baths with rising ethanol concentrations for 5 min in each bath, followed by bathing in Methyl tert-butyl ether (MTBE) plus epoxide mixture (in a 1:3 dilution) twice for 5 min each. Afterwards, the specimens were embedded in an epoxide mixture at 60°C for 48 h and then at 100°C for 11½ h. Semithin and ultrathin sections were cut on a Leica Microsystems microtome. Grids were purchased from Polyscience. The grids were then analyzed using electron microscopes (EM 109 and EM 902, Zeiss, Oberkochen, Germany) equipped with digital electron microscope cameras (Tröndle). 3 glomeruli from each mouse were analyzed.

Static histomorphometry was performed by the skelet.AL Skeletal Analysis Laboratories. Briefly, lumbar vertebrae 3 and 4 were fixed in 10% neutral buffered formalin, decalcified in EDTA and embedded in paraffin, and 3-μm sections were cut using a Leica Microsystems microtome (Leica Microsystems, Milton Keynes, UK). The sections were stained with either haematoxylin and eosin or tartrate-resistant acid phosphatise (TRAP) to identify osteoclasts and counterstained with Gill’s haematoxylin. The sections were examined by light microscopy (Leica Microsystems). The number of osteoblasts and osteoclasts per millimetre were measured on 6.5 mm of the corticoendosteal surfaces, starting 0.25 mm from the growth plate using the Osteomeasure analysis software (Osteometrics, Decatur, GA, USA).

Static histomorphometry was performed in paraffin embedded sections. Briefly, humorous and tibia fixed in 10% neutral buffered formalin, decalcified in EDTA (0.5 M) at pH 7.4, and embedded in paraffin, and 3 μm sections were cut using a Leica Microsystems microtome (Leica Microsystems, Milton Keynes, UK). The sections were stained with either haematoxylin and eosin or tartrate-resistant acid phosphatise (TRAP) to identify osteoclasts and counterstained with Gill’s haematoxylin. The sections were examined by light microscopy (Leica Microsystems). The number of osteoblasts and osteoclasts per millimetre were measured on 6.5 mm of the corticoendosteal surfaces, starting 0.25 mm from the growth plate using the Image J analysis software (Public Domain, BSD-2).