Anti cd24

Intestinal tissue was collected and fixed overnight in 4% PFA in PBS and embedded in paraffin. In all, 5-μm sections were stained by HE and PAS for histological analysis and goblet cells staining. Sample were embedded in OCT and 8-μm sections were performed for immunofluorescence staining by using the following primary antibodies: TUNEL (Roche, 11684817910), anti-Ki67 (BD; 550609), anti-CD24 (Thermo Fisher; MA5-11828), anti-Muc2 (Santa Cruz; sc-515032), anti-CgA (Immunostar; 20086). All primary antibodies were used as 1:100 dilutions. Goat anti-mouse Alexa fluor®488 (Abcam, ab150113) and goat anti-mouse Alexa fluor®647 (ab150115) secondary antibodies were used at 1:500–1:1000 dilutions.
For fluorescence in situ hybridizations (FISH), tissues of jejunum were fixed overnight in 4% PFA, paraffin embedded, and sectioned at 10 μm. The probe sequences targeting Lgr5, Olfm4 and Lys (Lysome) was: Lgr5 probe (5′-FAM-GACGACAGGCGGTTGGACGATAGGT-FAM-3′), Olfm4 probe (5′-FAM-CACTGACACCTCGCCACCATTCCA-FAM-3′) and Lys probe (5′-CY3-GCACCGATCATAGACCTTGGCCTGTA-3′). The protocols used for in vitro transcription and in situ hybridization were previously described^{44 (link)}. All images were acquired and processed with Zeiss Axio-Imager Z1 with apotome or Leica SP5 confocal microscope.

qRT-PCR was used to measure CD24 mRNA levels in IMR-32/WT, IMR-32/shRNA control (con), and IMR-32/CD24 shRNA cell samples. Samples were prepared as described above. TaqMan gene expression assays were used for CD24, normalized to GAPDH, and amplified in triplicate. The results are reported as the mean ± SEM.
Western blot analysis was also performed, using 2.5 × 10⁵ cells of each selected IMR-32 sample (as above). These cells were then boiled in NuPAGE LDS Sample Buffer (catalog no. NP0007, Thermo Fisher Scientific), and proteins were separated by electrophoresis on 4%–12% NuPAGE Bis-Tris denaturing polyacrylamide gels (catalog no. NP0321BOX, Thermo Fisher Scientific). Proteins were transferred to nitrocellulose membranes (0.2 µm, catalog no.1620112, Bio-Rad,) and probed with the following primary antibodies: anti-CD24 (Thermo Fisher Scientific, catalog n. MA5-11828, RRID:AB_10983158) at 1/200 and anti-GAPDH (catalog no. FL-335: sc-25778, Santa Cruz Biotechnology) at 1/2,000. Blots were probed with HRP-conjugated secondary antibodies (Invitrogen, catalog no. 62-6520, Goat anti-Rb, catalog no. 65-6120) and visualized using enhanced chemiluminescence chemiluminescence (catalog no. 32106, Pierce, Thermo Fisher Scientific).

CD19⁺ B cells were identified by flow cytometry after surface or intracellular staining with human-specific Abs conjugated with FITC, PE, APC, PE-cy7, PerCP-cy5.5, APC-cy7, BV421, or BV510. These human Abs included anti-CD3, anti-CD8, anti-CD19, anti-CD24, anti-CD25, anti-CD27, anti-CD40, anti-CD80, anti-CD86, anti-TGF-β, anti-TNF-α, anti-CCR3, anti-CXCR3, anti-IL-10, anti-IL-17, anti-IFN-γ, anti-Foxp3, anti-Viability Dye, and anti-IgD, which were purchased from Thermo Fisher (San Jose, CA, USA) or Thermo Fisher (Carlsbad, CA, USA). Appropriate species-matched Abs served as isotype controls. Flow cytometry was performed on a FACS Canto II (BD Biosciences), and data were analyzed using BD FCS Diva Software and FCS Express 5 plus software (De Novo Software, Los Angeles, CA, USA).

For stem and EMT features evaluation, 4-μm tissue sections were stained according to a well-established protocol in a Bond-RXm immunostainer (Leica, Munich, Germany). The following panels of primary antibodies were used: (1) Anti-panCK (1:10, clone AE1/AE3, Roche, Indianapolis, IN, USA), Anti-EpCam (1:500, clone E144, Abcam, Cambridge, UK), Anti-E-cadherin (1:10, clone EP700Y, CellMarque, USA), Anti-N-Cadherin (1:500, clone EPR1791-4, Abcam, UK), Anti-β4 integrin (1:400, clone JM11-06, Thermo, Waltham, MA, USA); (2) Anti-panCK (1:10, clone AE1/AE3, Roche, Indianapolis, IN, USA), Anti-CD24 (1:100, clone SN3b, Thermo), Anti-CD44 (1:100, clone 156-3C11, Thermo, USA), Anti-ALDH1 (1:500, polyclonal, Abcam, UK), Anti-β4 integrin (1:400, clone JM11-06, Thermo, USA). A 7-color Opal Kit (NEL797B001KT; PerkinElmer, Waltham, MA, USA) containing Opal 520, Opal 540, Opal 570, Opal 620, Opal 650, and Opal 690 fluorophores was used. The obtained slides were embedded in Mounting Medium with DAPI (VECTOR Laboratories Inc., Newark, CA, USA). The slides were scanned using Vectra 3.0 system (PerkinElmer, USA). Images were processed using inForm 2.2.1 software (PerkinElmer, USA). The frequency and the number of tumor cells in different populations as a percent of all tumor cells were assessed.

The following antibodies were used for FACS: FITC-anti-CD11b (11–0112), APC-anti-CD11b (17–0112), PE-anti-F4/80 (12–4801), FITC-anti-F4/80 (11–4801), APC-anti-CD45 (17–0451), PerCp-Cy5.5-anti-CD45 (45–0451), FITC-anti-CD24 (11–0242), APC-anti-CD24 (17–0242), PerCP-Cy5.5-anti-NK1.1 (45–5941), PE-anti-CD4 (12–0041), FITC-CD3e (11–0031), PerCP-Cy5.5-Gr1 (45–5931), and PE-anti-Ly6C (12–5932) obtained from eBioScience, and PE Annexin V Apoptosis Detection Kit I (559763) from BD Pharmingen. Cells were blocked for 15 min with Fc blocking reagent on ice prior to labeled with fluorescent-conjugated antibodies diluted in FACS buffer. Cells for phenotypic analyses were stained using the indicated fluorescent-conjugated antibody for 30 min on ice. Appropriate isotype controls were used in all case. Flow cytometry was performed using a FACSCalibur (BD Bioscience, San Jose, CA) and the data were analyzed with FlowJo software (TreeStar, Ashland, OR).