Dried specimens are mounted on a SEM stub with double-sided tape and covered with a thin layer of gold with a sputter coater. The fractured surfaces of the kidney are viewed on a scanning electron microscope. Fractures tend to follow voids and weakness in the frozen tissue and should reveal primary cilia within the tubule (Fig. 2), duct and Bowman’s capsule. In the healthy adult kidney primary cilia are often obscured

within the proximal tubule brush border. Segments of the collecting duct are recognizable by the presence of intercalated cells which do not bear a primary cilium.[11] SEM can also be used to examine apical primary cilia on Ixazomib cultured cells as described above, but without the need for cryoprotection and freeze fracture. Fluorescence microscopy is the technique of choice for most studies of renal primary cilia. An advantage of this approach is the availability of antibodies (Table 1). Transgenic cell lines have also been used to study the dynamics of ciliary components in cultured renal cells

as described elsewhere.[27] Sample preparation protocols for fluorescence microscopy vary depending Obeticholic Acid ic50 on the nature of the specimen (cultured cells or kidney section), the antibodies being used and the antigens being localized. Clone 6-11B-1 Cat no. T6793 Antibody N-18 Cat no. sc-49459 Santa Cruz Biotechnology Rodent kidneys are prepared for immunofluorescence by fixing in 4% formaldehyde

in PBS. Best preservation is achieved by initially perfusion fixing using the procedure described for electron microscopy, Lepirudin then immersion fixing of pieces of kidney for 2–5 h at room temperature. Human kidney samples can be immersion fixed with 4% formaldehyde, although renal biopsy samples are often fixed with formalin for pathology which is also acceptable for cilium immunostaining. Glutaraldehyde is generally avoided for tissue destined for fluorescence microscopy as it increases autofluorescence, particularly of tubules in the kidney. For sectioning, fixed kidney is embedded in paraffin or frozen. Paraffin sections cut at approximately 6 microns are baked at 60°C for 1 h, dewaxed in xylene and rehydrated through decreasing ethanol concentrations, water and then PBS. Paraffin-embedded samples require antigen retrieval by proteinase K digestion (20 μg/mL in TE for 10 min at 37°C) or boiling in citrate buffer (10 mM sodium citrate, pH 6). In our experience, boiling citrate buffer gives clearer cilium labelling in the kidney using anti-acetylated alpha-tubulin, and also works well for human renal biopsy samples fixed in formalin and embedded in paraffin[5] (Fig. 3a). However, antigen retrieval methods can be varied to optimize the detection of other antigens with respect to primary cilia.