Tritc

HCC tissues were fixed with 10% neutral formalin at room temperature for 24 h. After dehydration, transparency, and paraffin embedding, serial paraffin sections (4 µm thick) were generated, followed by deparaffinization and rehydration. Sections were incubated with anti-Hep Par-1 and CK19 antibodies simultaneously. Then, sections were stained with goat anti-rabbit secondary antibody labeled with tetramethylrhodamine-5(6)-isothiocyanate (TRITC; 111-026-045, Jackson ImmunoResearch, USA) and goat anti-mouse secondary antibody labeled with fluorescein isothiocyanate (FITC; 111-026-045, Jackson ImmunoResearch, USA) under dark conditions at 37 °C for 1 h. Cells were then counterstained with 4',6-diamidino-2-phenylindole (DAPI; C0065, Solarbio, China) for 2 min to localize the nuclei. Finally, sections were viewed under a fluorescence microscope.

Testes collected from Kit^W/Kit^WV mice 2 months after SSC transplantation were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Tissue sections (5 μm in thickness) were obtained in a microtome and mounted on glass slides. Sections were dewaxed and rehydrated, followed by antigen retrieval in 10 mM sodium citrate buffer. After blocking with 5% BSA in PBS (wt/vol) for 1 h, the sections were incubated with primary antibody at 4 °C overnight (see Table 1). Secondary antibody conjugated with either FITC or TRITC (Jackson ImmunoResearch) at 1:200 dilution was used and incubated for an hour at room temperature. Slides were then stained with DAPI (blue) to visualize cell nuclei and mounted in prolonged anti-fade mounting medium. Fluorescence images were captured using a Zeiss LSM780 confocal microscope.
Table 1

Antibodies used in this study

Antibody	Vendor	Catalog no.	Host	Working dilution
Tra98	Abcam	Ab82527	Rat	1:200
GFRα-1	R&D	AF560	Goat	1:200
C-kit	Abcam	Ab5506	Rabbit	1:200
SCP3	Abcam	Ab97672	Mouse	1:200
N-cadherin	Santa Cruz	sc-7939	Rabbit	1:200
JAM-A	Thermo	36-1700	Rabbit	1:200
Lin28a	R&D	AF3757	Goat	1:200
SOX9	Abcam	ab76997	Mouse	1:200

Abcam, Cambridge, UK; Santa Cruz Biotechnology, Dallas, TX, USA; Thermo Fisher Scientific, Waltham, MA, USA; R&D Systems, Minneapolis, MN, USA

Cells were fixed in either 4% PFA for 20 min, in ice-cold methanol for 10 min (for γ-tubulin and centrin antibodies) or for 10 min in 1× PTEMF extraction buffer (20 mM PIPES, pH 6.8, 10 mM EGTA, 1 mM MgCl 2, 0.2% Triton X-100 and 4% PFA) prepared fresh from 4× stock solution (for all the mitotic checkpoint antibodies). Cells were permeabilized by 2×3 min wash in 0.1% Triton-X-PBS (PBS-TX), followed by blocking in PBS-TX containing 1% BSA for 30 min at room temperature, before being processed for immunofluorescence. Primary antibodies were diluted in 1% BSA in 1× PBS-TX in a 37°C incubator for 1 hr. Primary antibody dilutions were as follows: mouse anti-α-tubulin (DM1α, Sigma, 1∶1000), rabbit anti-γ-tubulin (Sigma, 1∶1000), rabbit anti-pericentrin (Bethyl, 1∶300), mouse anti-centrin-3 (Abnova, 1∶500), rabbit anti-TPX2 (Bethyl, 1∶100), mouse anti-Hec1 (GeneTex, 1∶100), human anti-centromere antibody (ACA, 1∶2000), mouse anti-BubR1 (Millipore, 1∶50), mouse anti-Mad1 (a kind gift from Dr. Andrea Musacchio, 1∶300), sheep anti-Mad2 (a kind gift from Dr. Steven Taylor's Laboratory, 1∶200), rabbit anti-Zwint-1 (Bethyl, 1∶100). Secondary antibodies conjugated to FITC, TRITC or Cy-5 (all from Jackson Laboratories) were chosen as appropriate and used as recommended by the supplier. DNA was counterstained with DAPI.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100/PBS for 5 min. After blocking with 2% FBS in PBS, cells were incubated with the primary antibodies against IgG, CK18, vimentin, or microtubulin for 1 h at room temperature. Secondary antibodies were goat anti-mouse or anti-rabbit labeled with Alexa 488 or TRITC (Jackson ImmunoResearch Laboratories, Inc.) for 1 h. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (Sigma). Confocal images were acquired with a TCS S2 microscope adapted to a DMIRBE inverted microscope (Leica Microsystems, Inc.).

Intracellular TG2 transamidase activity was measured as previously described [34 (link)]. Briefly, the cells were seeded in Greiner 96-well microtiter plates (Greiner Bio-One, Kremsmuenter, Austria) and incubated with 0.2 mM 5BAPA and 0.1 mM aminoguanidine overnight at 37 °C. The cells were treated with increasing concentrations of ACR in serum-free media for 4 h and then fixed with a 10% formaldehyde solution, permeabilized, blocked, and stained with streptavidin-tetramethylrhodamine isothiocyanate (TRITC, Jackson ImmunoResearch Laboratories). Intracellular TG2 transamidase activity was then detected as a fluorescence signal from TRITC and analyzed using an ImageXpressMICRO High Content Screening System (Molecular Devices, Sunnyvale, CA, USA). Morphological analysis was performed using a Multi-Wavelength Cell Scoring Application Module in MetaXpress Image Analysis software (Molecular Devices).

Cells were incubated with 5% donkey serum (Jackson ImmunoResearch Laboratories) in PBST (PBS + 0.1% Triton-X) for 30 min to avoid nonspecific antibody binding and permeabilize the cells. Primary antibodies, diluted in 5% donkey serum/PBST, were then incubated at 4 °C overnight with shaking. After primary antibody incubation, cells were washed three times with PBST and donkey secondary antibodies conjugated with Alexa Fluor 488 (i.e., Anti-Goat, Anti-Rabbit, Anti-Chicken, Anti-Mouse) or TRITC (i.e., Anti-Goat, Anti-Rabbit, Anti-Chicken, Anti-Mouse, Anti-Rat) (Jackson ImmunoResearch Laboratories) diluted at 1:400 in 5% donkey serum/PBST were added to the cells for 30 min at RT. Then, cells were thrice washed with PBST and nuclei were stained with DAPI (Roche Diagnostics). Antibody sources, catalog numbers, and dilutions are listed in Supplementary Table 2. For imaging, we used Leica DMI6000 or confocal Leica TCS SPE. Images were initially processed by LAS X software and then further analyzed and quantified using ImageJ software (NIH, W Rasband, http://rsb.info.nih.gov/ij) using cell counter plug-in. Typically, at least five randomly selected images were counted per condition and per replicate.