Anti cp110

Immunohistochemical staining of Ki67 and CP110 was performed as described previously47 (link). Briefly, paraffin-embedded 4 μm-thick sections were deparaffinized, heated in citrate buffer (0.01 M), treated with 0.3% H₂O₂ (v/v), and re-hydrated. After blocking, rabbit polyclonal anti-CP110 (1:100, ProteinTech Group, Chicago, IL, USA) and anti-Ki67 (1:50, ProteinTech Group, Chicago, IL, USA) primary antibodies incubated overnight at 4 °C. After rinsing in PBS, slides were incubated for 25 min at room temperature with biotinylated goat-anti-rabbit immunoglobulin followed by incubation with peroxidase-conjugated streptavidin for 20 min and fresh 0.05% 3, 3′-diaminobenzidine (DAB). Slides were counterstained with Mayer’s hematoxylin and mounted with crystal mount. The methods were carried out in accordance with the approved guidelines and all experimental protocol were approved by a named institutional and/or licensing committee.

Embryos were fixed with MEMFA or Dent’s fixative solution overnight at 4°C, and RPE1 cells were fixed with 4% PFA solution for 20 min at room temperature. Fixed embryos or RPE1 cells were incubated in a blocking solution (10% FBS and 2% DMSO in 1×TBS with 0.1% Triton X-100) for 30 min at room temperature to block non-specific binding. Immunostaining was performed with the following antibodies at 1:50–500 dilutions in blocking solution for 1–2 hr at room temperature: anti-DDDD-K (Abcam, Cambridge, UK, ab1162), anti-GFP (ThermoFisher Scientific, A10262), anti-HA (Santa Cruz, sc-7392), anti-RFP (Abcam, ab62341), anti-acetylated tubulin (Sigma Aldrich, T7451), anti-γ-tubulin (Abcam, ab191114), anti-MyHC (DSHB, IA, USA, A4.1025), anti-CP110 (Proteintech, IL, USA, 12780–1-AP), anti-BBS4 (Proteintech, 12766–1-AP), anti-TGN46 (Abcam, ab50595), anti-Arl13b (Proteintech, 17711–1-AP), anti-TTBK2 (Proteintech, 15072–1-AP), anti-IFT20 (Proteintech, 13615–1-AP), anti-IFT88 (Proteintech, 13967–1-AP), anti-Rab8a (Cell Signaling, MA, USA, #6975), anti-Rab11 (Cell Signaling, #5589), and anti-GJA1 (both ThermoFisher Scientific, PA1-25098, 13–8300). Fluorescence labeling was performed using DAPI (1:10,000, ThermoFisher Scientific, D1306), and Alexa Fluor 488-, 555-, and 633-conjugated secondary antibodies (1:300, all ThermoFisher Scientific).