Ab89901

The transplanted tumors were surgically removed until the tumor volume reached 100 mm³, and then fixed in 10% formalin. Then, the tumors derived from the donor’s pelvic lymph node and the transplanted tumors were subjected to routine hematoxylin and eosin (HE) staining and staining for other markers, including P16 (1:50, ab51243, Abcam, Cambridge, UK), P53 (1:2000, ab32389, Abcam), ER (1:800, 21244-1-AP, Proteintech), PR (1:200, 25871-1-AP, Proteintech, Chicago, IL, USA), PAX8 (1:200, 10336-1-AP, Proteintech), and WT1 (1:500, ab89901, Abcam). Microscopic slides were reviewed by a senior gynecology-dedicated pathologist (Prof. Yang).
Similarly, immunohistochemistry (IHC) was conducted to assess the expression of Ki67 (1:200, AF0198, Affinity, San Francisco, CA, USA), CyclinD1 (1:250, ab134175, Abcam), Hes1 (1:200, ab108937, Abcam), N-cadherin (1:200, #13116, Cell Signaling Technology, Danvers, MA, USA), vimentin (1:200, #5741, Cell Signaling Technology) and CSF3 (1:200, 17185-1-AP, Proteintech) in the tumors extracted from mice. The kidney and liver were also removed for HE staining to evaluate the toxicity of viscera.
Finally, the expression of CSF3 in the corresponding lymph nodes samples from 10 ovarian cancer patients was evaluated by IHC. The scoring criteria for staining intensity were described in our previous research [33 (link)].

Tissue and PDTO were fixed in 3% paraformaldehyde overnight. After embedding PDTO in 2% agarose, tissue and PDTO were dehydrated, paraffin embedded, and sectioned before standard hematoxylin and eosin staining (H&E). Automated immunohistochemistry using a Ventana Discovery XT autostainer (Roche) was performed on 4 µm-thick paraffin sections. Slides were deparaffinized with EZPrep buffer and epitopes were unmasked by 15 min of high-temperature treatment in CC1 EDTA buffer. Sections were incubated for 40 min at 37°C with an anti PAX8 (ab191870, Abcam, 1/500), p53 (ab16665, Abcam, 1/100), WT1 (ab89901, Abcam, 1/300), HNF1β (ab213149, Abcam, 1/2000), Ki67 antibody (NCL-Ki67p, Novocastra, 1/500) or Napsin A (ab133249, Abcam, 1/2000). Secondary antibody (Omnimap Rabbit HRP; Ventana Medical System Inc., Tucson, AZ, USA) was incubated for 16 min at room temperature. Immunodetection performed without the primary antibody was used as control. After washes, the staining was performed with DAB (3, 3'-diaminobenzidine) and sections were counterstained with hematoxylin using Ventana reagents according to the manufacturer's protocol. Stained slides were then digitized using an Aperio ScanScope slide scanner (Aperio Technologies).

Kidney biopsies and 293 T cells fixed in neutral buffered formalin were embedded in paraffin or optimal cutting temperature compound by using standard procedures. Frozen and paraffin sections were stained with immunofluorescence, respectively. Immunofluorescent staining and images were obtained by a Nikon A1R Meta confocal microscope. Cover slips were observed.
The antibodies used were list below: anti-Cubilin-C-terminal antibody (1:500, ab191073, Abcam), Rat Cubilin(CUBN) polyclonal antibody (1:100, 31010, Bicell Scientific), anti-Synaptop-odin antibody (1:50, 21064-1-AP, Proteintech), anti-Wilms Tumor Protein antibody (1:50, ab89901, Abcam), anti-COL4A3 antibody (1:100, Kingmed, Guangzhou, China), anti-COL4A5 antibody (1:100, Kingmed, Guangzhou, China), anti-Amnionless antibody (1:10, sc-365384, Santa Cruz), anti-Megalin Antibody (1:30, CD7D5, Novus Biologicals), goat polyclonal secondary antibody to mouse Alexa fluor 488 (1:400, ab150113, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 555 (1:400, ab150078, Abcam), rabbit monoclonal to HA tag (1:500, ab236632, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 647 (1: 400, ab150079, Abcam), goat anti-mouse Alexa fluor 568 (1: 400, ab175473, Abcam), DAPI (1:1000, C1002, Beyotime).

Total protein from the kidney tissues was isolated after being lysed by RIPA buffer (01408, Beyotime Biotechnology, China) containing PMSF, protease, and protein phosphatase inhibitor for 30 min on ice. All samples were centrifuged at 12000 g for 15 min (4°C). Protein concentration was determined using the BCA assay kit (P0012, Beyotime Biotechnology, China). An equal amount of protein (40 μg) was subjected to 8–10% SDS-PAGE gel and then transferred to PVDF membranes (162–0177, BIO-RAD, USA). The membranes were blocked with 5% BSA and incubated overnight with the following antibodies at 4°C: antinephrin (ab216341, Abcam, Cambridge, UK), anti-Wilms Tumor 1 (WT1) (ab89901, Abcam, Cambridge, UK), anti-Aquaporin 1 (ab168387, Abcam, Cambridge, UK), and anti-GAPDH (EM1101, Huaan Biotechnology, Hangzhou, China). After incubating with peroxidase-conjugated secondary antibodies (goat anti-rabbit-antibody, LI-COR Biosciences, USA), the protein expression bands were developed with chemiluminescence. The densitometry of the brands was then calculated using the Odyssey near-infrared dual-color laser imaging system (Odyssey Clx, LI-COR Biosciences, USA).