Calretinin

Cells harvested from pleural effusions were resuspended in 1–3 drops of sheep plasma. A drop of thrombin (0.5 mL thrombin plus 9.5 mL 0.1 M CaCl₂) was added to make a solid pellet, which was fixed with 10% formalin, processed with graded concentrations of ethanol and isopropanol, and then embedded in paraffin. Sections were cut 4 μm thick, deparaffinised, and rehydrated prior to quenching with 1% H₂O₂. AQP1 immunohistochemistry was performed as previously described [21 (link)]. Calretinin and CAM5.2 immunohistochemistry was performed with citric acid retrieval prior to the addition of primary antibody (Calretinin; 1/5000; Invitrogen, CAM5.2, 1/1000; BD Australia). Both methods utilized the Novo Link polymer (Leica Microsystems, Wetzlar, Germany) and DAB + Chromogen (Dako, CA, USA) detection systems prior to hematoxylin counterstaining.

Histological examination, whole-mount and section immunostaining and ISH were carried out according to standard procedures. Briefly, inner ears were fixed in 4% paraformaldehyde (PFA) for 1 hr at 4°C, dehydrated, and embedded in wax. Paraffin sections were generated at 6 μm. For ISH, tissues were fixed overnight. We used five embryos for each genotype at each stage for each probe and the result was consistent in each embryo.
Primary antibodies: anti-Sox2 (PA1-094, Thermo Fisher), -Myo7A (25–6790, Proteus and 138-1-s, DSHB), -Six1 (HPA001893, Sigma), -Atoh1 (Math1-s, DSHB), -p27^kip1 (554069, BD Pharmingen), -Calretinin (MA5-14540, Thermo Fisher), -p75^NTR (#07–476, EMD Millipore), -N-cadherin (610921, BD Bioscience), -E-cadherin (U3254, Sigma), -S100A (ab11428, Abcam), -GLAST (ab416, Abcam), -Pou4f3 (sc-81980, Santa Cruz), -Prox1 (AB5475, Millipore), -Acetylated tubulin (T7451, Sigma), -Cy3-, Cy2-, Cy5- and FITC-conjugated secondary antibodies were used. Alexa Fluor 488 or 350-conjugated phalloidin (A12379 and A22281, Life technologies) were used for actin staining. Hoechst 3342 was used for nuclear staining.

The mesothelial features of cultures were confirmed by immunohistochemical (IHC) staining carried out on cells at passage 1. Specifically, cells were centrifuged at 1200× g for 5 min, fixed overnight in 4% v/v formalin at 4 °C, and then paraffin-embedded. The following antibodies were used: BAP-1 (Santa-Cruz Biotechnology, Santa Cruz, CA, USA, sc-28383, 1:100); Pan-cytokeratin AE1/AE3 (Dako, Agilent, Santa Clara, CA, USA, GA053, 1:500); Wilms Tumor-1 antigen (WT1) cl.6FH2 (Thermo Fisher Scientific, Waltham, MA, USA, MA1-46028, 1:10); Calretinin (Thermo Fisher Scientific, RB-9002-R7, 1:100). Mesothelial origin was confirmed if positivity for at least one between Calretinin and WT1 was detected, as well as in the case of positivity for pancytokeratin. The histological features are reported in Table 1.