Cryosections are useful for difficult antigen–antibody combinations because antigenicity is maintained better than in paraffin. This comes at the cost of reduced structural detail, but cilia are still preserved (Fig. 3b). Frozen sections are thawed and dried at room temperature then rehydrated in PBS and labelled without antigen retrieval. Further fixing in formaldehyde prior to labelling may help preserve details of cilia and associated structures in delicate or lightly fixed sections. Treating sections with 0.1% Triton X-100 or other detergents can improve staining by increasing antibody access. For immunostaining primary cilia in culture (Fig. 3c–f), cells are

typically grown as a monolayer on a coverslip and starved of serum for 24 h R788 purchase to induce cell cycle exit and ciliogenesis. Cultures click here are fixed for 5 min with 2–4% formaldehyde and permeablized with 0.1% Triton X-100 in PBS for 5–15 min. If this approach does not give good immunolabelling for a particular antigen an alternative is to fix and permeablize/extract with ice cold methanol, dry for 5 min at room

temp, then rehydrate with PBS. Table 1 details commercially available antibodies that label the renal primary cilium and relevant references including published examples of their use. Standard indirect immunostaining protocols are used with primary antibodies against ciliary components being detected by fluorochrome conjugated secondary antibodies. Primary cilia are small and it is important that immunostaining protocols are optimized to allow their detection. Non-specific antibody binding is blocked using bovine

serum albumin, compatible serum, or commercially available blocking solutions. If a mouse antibody is used on mouse kidney, immunoglobulin blocking steps (e.g. Vector laboratories MOM kit) are used to prevent the secondary antibody recognizing endogenous mouse Atazanavir immunoglobulins in the sample. Optimal antibody dilutions should be obtained from previous publications or determined empirically to give the best signal to noise ratio. Including 0.05% Tween-20 detergent in antibody dilutions and washes may reduce nonspecific background. Isotype and single antibody (in the case of double labelling) control experiments should be performed to confirm the specificity of primary cilium labelling, and to verify that filter sets and fluorochromes used give an unambiguous signal in the expected channel. For labelling the axoneme of the primary cilium, mouse monoclonal anti-acetylated alpha-tubulin is a reliable and widely used option. This antibody was raised against acetylated alpha-tubulin from the sea urchin sperm axoneme, and specifically recognizes this modified form of tubulin in a diverse range of species.[45] The tubulin in more stable microtubules becomes acetylated meaning that the microtubular cytoskeleton of the cilium is preferentially labelled compared with microtubules of a more transient nature in the cytoplasm (Fig. 3a–c,e).