Rabbit monoclonal anti-acetylated tubulin Torin 1 is also available and in our experience gives the same pattern of labelling as the mouse version. Other antibodies against alpha- and beta-tubulin will label the cilium, but the signal from the cilium may be lost among other structures containing tubulin, particularly in sections of a complicated organ such as the kidney. Arl13b is a small GTPase that is defective in Joubert syndrome, a ciliopathy with a cystic renal phenotype. Arl13b is associated with the ciliary membrane and antibodies against this protein reliably label primary cilia (Fig. 3d–f)
in the kidney and in cultures of renal epithelial cells.[48-50] Labelling of the renal primary cilium using rabbit polyclonal anti-Arl13b or rabbit monoclonal anti-acetylated tubulin are useful approaches when co-labelling with a mouse monoclonal antibody against another ciliary or marker protein precludes the use of mouse monoclonal anti-acetylated tubulin. Gamma-tubulin is a component of microtubule organizing centres and is found in the region of the basal body.[57-59] Antibodies against this tubulin check details isoform can be used to determine the orientation of cilia labelled with anti-acetylated tubulin. In this case a rabbit polyclonal anti-gamma-tubulin is used to label the basal body in combination with
mouse monoclonal anti-acetylated alpha-tubulin labelling of the axoneme (Fig. 3b). The basal body is an essential staging area required for the assembly and normal function of the cilium so anti gamma-tubulin is used to assess basal body
localization of cilium-associated transport and signalling components. Monoclonal mouse anti-gamma-tubulin is also available and can be used in combination with polyclonal antibodies from other species. Several proteins that are defective or deficient in human and/or animal models of cystic kidney disease have also been immunolocalized to the primary cilium and basal body. These proteins include MKS1, Nephrocystins, BBS proteins and IFT components such as IFT88 (Table 1). The key human PKD proteins polycystin-1, polycystin-2 and fibrocystin are difficult to raise effective antibodies against. Commercially available antibodies are useful for immunoblotting, but published examples BCKDHB of immunolocalization to the primary cilium typically use antibodies produced by the authors or generous colleagues.[15, 46, 60-62] Nuclear counterstains for DNA (DAPI or Hoechst) and segment/cell type specific markers compliment primary cilium immunolabelling and facilitate navigation within the kidney (Fig. 3). Useful markers include: Lotus tetragonolobus lectin for the proximal tubule (Fig. 3a), anti-thiazide-sensitive sodium chloride cotransporter for the distal tubule, and Dolichos biflorus lectin for the collecting duct.