Rm 9106 s1

IHC was performed with antibodies directed against B220/CD45R (RA3-6B2; R&D Systems; 1:40 dilution), CD138 (281-2; BD Biosciences; 1:50 dilution), CD3 (A0452; DAKO; 1:100 dilution), myeloperoxidase (A0398; DAKO; 1:100 dilution), Ki-67 (RM-9106-S1; Thermo Fisher Scientific; 1:200), Bcl6 (sc-858; Santa Cruz Biotechnology; 1:50), Irf4 (sc-6059; Santa Cruz Biotechnology; 1:100), and Pten (M362729-2; Agilent; 1:150 dilution). Pre-treatment of sections was conducted with EDTA for 20 min (B220, CD138, CD3, myeloperoxidase), 30 min (Ki-67, Irf4, Pten), or 40 min (Bcl6).
We used goat anti-rabbit, rabbit anti-rat, rabbit anti-goat, and rabbit anti-mouse secondary antibodies (AffiniPure Goat Anti-Rabbit IgG (H+L) [111-005-003; Jackson ImmunoResearch], AffiniPure Rabbit Anti-Rat IgG [312-005-045; Jackson ImmunoResearch], Rabbit Anti-Goat IgG [P0449; DAKO], and Rabbit Anti-Mouse IgG [ab125904; Abcam]).

Expression levels of the cellular proliferation marker Ki-67 in the skin were determined. Briefly, after the deparaffinized sections were subjected to heat-induced antigen retrieval in antigen retrieval solution (citrate buffer, pH 6.0) at 121 °C for 10 min, the sections were washed with water, incubated with 3% H₂O₂ solution for 5 min to block endogenous peroxidase and washed three times for 5 min each with 0.01 M PBS (pH 7.4). Then, the sections were incubated with anti-rabbit Ki-67 (RM-9106-S1, Thermo Fisher Scientific Inc., Waltham, MA, USA) as a primary antibody for 50 min at room temperature and washed three times for 5 min each with PBS. To detect Ki-67 expression, the sections were further incubated with Histostar™ (Rb) for Mouse tissue (8470, Medical & Biological Laboratories Co., Ltd., Aichi, Japan) for 30 min at room temperature and washed three times with the same buffer for 5 min. Ki-67 expression in the sections was detected using 3,3′-diaminobenzidine solution as a chromogen substrate and counterstained with hematoxylin. Three different microscopic fields per plate were photographed, and Ki-67-positive cells in the skin were counted.

The esophagus tissues were fixed in 10% buffered formalin for 48 hours. The paraffin-embedded 5 μm-thick esophagus tissue sections from different groups were processed for H&E staining. For IHC staining, slides were incubated at 55°C for 2 hours and deparaffinized in xylene and rehydrated through graded alcohols (100%, 95%, 70% alcohol; 2 times 3 minutes in each grade). Slides were rinsed in ddH₂O and then blocked with 0.3% H₂O₂ for 30 minutes to block endogenous peroxidase activity. Heat-induced antigen retrieval was performed in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) using a pressure cooker. Sections were rinsed in PBS and blocked in 10% normal donkey serum, 1% bovine serum albumin and 0.1% Triton X-100. Sections were incubated with primary antibody against Keratin 5 (Abcam, ab52635, 1:500), Mcl1 (Abcam, ab32087, 1:200), p-p65 (Abcam, ab86299, 1:100), caspase3 (Cell Signaling, #9663, 1:500) and Ki67 (Thermo Fisher Scientific, RM-9106-S1, 1:200) overnight at 4°C. Section were rinsed in PBS and incubated with biotinylated secondary antibody and streptavidin-biotinylated horseradish peroxidase complex (Zhongshan, Beijing, China) 1 hour at room temperature. Proteins expression was detected by using Diaminiobenzidine (DAB) as substrate.

Mice bearing orthotopic xenografts generated as described above were treated with vehicle (both Tween/PEG and 5% dextrose via i.p. injection Monday, Wednesday and Friday for a total of 12 treatments and sacrificed 24 hours after the last dose. Mouse brains were harvested and following formalin fixation were sectioned in a mouse brain cutter into either coronal or sagittal sections. Processing of tissue was performed in the Histology Unit at the Garvan Institute of Medical Research. Morphologic assessment of xenografts is based on routine haematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining with Vimentin (1:400; Novocastra NCL-VIM-V9) and Ki67 (1:400; Thermoscientific; RM-9106S1). Representative photographs of a vehicle control xenograft (shown in Fig 6B) were taken using an Olympus BX53 light microscope and CD-73 camera with cellSens software.