Prb 160p

Thymuses were dissected and fixed overnight in 10% neutral buffered formalin, transferred to 70% ethanol, and routinely processed and embedded in paraffin. Four μm sections were stained with haematoxylin and eosin (H.E.) for histopathological examination. Immunohistochemistry (IHC) and immunofluorescence (IF) analyses were performed as previously described [14 (link)]. Antibodies included: anti-K8/18 (1:500, Guinea Pig polyclonal, GP11, Progen Biotechnik, GMBH, Heidelberg, Germany), anti-K19 (1:500, Rabbit monoclonal, Epitomics, CA), anti-K5 (1:3000, Rabbit polyclonal, PRB-160P, BioLegend, Dedham, MA), anti-GFP (1:200, monoclonal, b-2, Santa Cruz), anti-Ki67 (1∶500, rabbit polyclonal, 06–570, BD Pharmingen, San Diego, CA), and anti-SV40 T antigen (1:100, mouse monoclonal, DP02-200UG, Calbiochem). For double or triple IF staining, the first primary antibody (anti-K8/18) was incubated for 2 hours at room temperature followed by the second and third primary antibody (anti-GFP, anti-K5, anti-Ki67, and/or anti-T₁₂₁) incubation overnight. Mixed Alexa fluor 488, 594, and 633 (1:200 dilution, Invitrogen) served as secondary antibodies. Nuclei were stained with DAPI. Images were captured using Zeiss light, immunofluorescence, or confocal microscopes.

Tissues sections were obtained and analysed as previously described [27 (link)]. The following antibodies were used: cytokeratin 5 (1:500, PRB-160P; BioLegend, San Diego, CA), cytokeratin 14 (1:500, PRB-155P; BioLegend), cytokeratin 8 (1:500, MMS-162P; Biolegend), p63 (1:100, sc-8431, clone 4A4; Santa Cruz Biotechnologies, Santa Cruz, CA), integrin β1 (10 mg/ml, MAB1997, clone MB1.2, Chemicon, Temecula, CA), Alexa Fluor 594 phalloidin (1:100, Molecular Probes, Eugene, OR), alpha smooth muscle actin–cy3 (1:400, C6198, Sigma), Wif1 (1:100, ab33281, abcam, Paris, France). Secondary antibodies were from Molecular Probe: Alexa Fluor 594 F(ab’)2 fragment of goat anti-rabbit IgG (1:1000, A11072), Alexa Fluor 594 F(ab’)2 fragment of goat anti-mouse IgG (1:1000, A11020), Alexa Fluor 594 F(ab’)2 fragment of goat anti-Rat IgG (1:1000, A11007). Pictures were taken either on a LSM 510 META Confocal Microscope or an Axioplan2 with ApoTome equipment (Carl Zeiss, Germany).

Tissue lysates were collected using whole cell lysis buffer together with a homogenizer. Whole cell lysis buffer contains 20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM sodium fluoride, 0.5 mM sodium vanadate, 1 mM sodium pyrophosphate, 0.5 mM 4-(2-aminmoethyl) benzensulfonyl fluoride hydrochloride and protease inhibitors, and protease inhibitors were freshly added into the whole cell lysis buffer immediately prior to protein collection^{52 (link)}. Total protein concentration of tissue lysates was determined using bicinchoninic acid assay and a microplate reader following the manufacturer’s instructions. The same amount of tissue lysates denatured at 95 °C for 5 min were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and then transferred onto polyvinylidene difluoride (PVDF) membranes. Blocking was performed with 5% milk. PVDF membranes were incubated with primary antibodies. Antibodies against Krt5 (#PRB-160P, 1:2000), Krt6 (#PRB-169P, 1:2000), and Lor (#905104, 1:1000) were obtained from BioLegend, and α-tubulin (#NB600-506, 1:5000) from Novus. The signal was detected using peroxidase-conjugated secondary antibodies and an enhanced chemiluminescent horseradish peroxidase substrate. The intensity of signal was determined using ImageJ.