Streptavidin horseradish peroxidase

The protocol employed was based on that previously described (14 (link), 24 (link), 27 (link)), with minor modifications. Briefly, polystyrene microplates were coated ON at 4°C with 0.1 ug of purified ZnT8/GAD65 chimera per well. After washing with PBS, adding 200 μL of blocking solution per well for 1.5 h, and washing five times with PBS-T, 50 ul samples were added in duplicate. After 1 h of incubation at room temperature, plates were washed again and 56.0 ng of ZnT8/GAD65-biotin per well were added. One hour later, another round of washing steps was done, and the bound ZnT8/GAD65-biotin was detected by the addition of Streptavidin-Horseradish Peroxidase (Jackson ImmunoResearch Laboratories, Inc.). Finally, microplates were washed five times with PBS-T plus one final washing step with PBS. The color reaction was elicited by the addition of the chromogenic substrate incubated for 15 minutes in the dark and was then stopped with 4N H₂SO₄. The oxidized substrate was measured at 450 nm with an ELISA plate reader. The blank control was made by replacing serum samples with PBS‐MT. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs = (A‐Ac)/SDc, where Ac is the mean specific absorbance of 25 healthy control sera and SDc its standard deviation. The cut‐off value of the assay was set at 2 SDs.

Intestinal tissue was paraffin-embedded and cut into five-micron-thick longitudinal sections followed by H&E staining. For immunohistochemistry, the un-stained slides were deparaffinized and blocked with 3% hydrogen peroxide in methanol. Antigen retrieval was performed using Diva Decloaking solution (Biocare Medical, Concord CA) (120 °C for 2 min using a pressure cooker). Slides were blocked with avidin-pink and biotin-blue (Biocare Medical), treated with anti-DCLK1 antibody (#62257, 1:400; Cell Signaling Technology, Danvers MA) in DaVinci Green (Biocare Medical), incubated overnight at 4 °C, and visualized with biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch Inc. West Grove PA) followed by streptavidin-horseradish peroxidase (Jackson ImmunoResearch Inc. West Grove PA), diaminobenzidine (Sigma-Aldrich), and hematoxylin counterstaining. Organoid whole-mount immunofluorescence staining was performed according to published method^{17 (link)} with minor modification. Briefly, the organoid was fixed with 10% neutral-buffered formalin, quenched with 50 mM NH₄Cl, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, treated with DCLK1 antibody (#62257, 1:100; Cell Signaling Technology, Danvers MA) and finally visualized with Alexa Fluor^® 488 Conjugate anti-rabbit secondary antibody (#4412, 1:250; Cell Signaling Technology, Danvers MA).

GeXIVA[1,2] (TCRSSGRYCRSPYDRRRRYCRRITDACV; MW = 3451.3 Da; purity: 97.67%; Lot: P483937-2-Y) was provided by GL Biochem Ltd. (Shanghai, China). The bovine albumin (BSA), casein, Rapid Equilibrium Dialysis (RED) Device Inserts, 8K MWCO, and Baseplate were purchased from Thermo Fisher (San Jose, CA, USA). The cOmplete^TM proteinase inhibitor was purchased from Roche (Basel, Switzerland). Phosphate buffered saline (PBS), PBS with 0.05% Tween 20 (PBST), 3,3′,5,5′-Tetramethylbenzidine (TMB), and stop solution were purchased from Solarbio (Beijing, China). The GeXIVA[1,2]-specific antibody 4B2 and biotin-2# were prepared in our laboratory. Streptavidin-Horseradish Peroxidase was purchased from Jackson ImmunoResearch (West Grove, PA, USA)

TME-stimulated and vehicle-treated MCF-7 and T47D cells were grown (as described above) for different time points (as described in Figure legends). Then, conditioned media (CM) were removed and CXCL8, IL-6, IFNβ or IFNγ levels were determined by ELISA using standard curves at the linear range of absorbance. To this end, recombinant human CXCL8 (rhCXCL8; #200-08; PeproTech) or rhIFNβ (#300-O2BC; Peprotech) were used. Coating polyclonal antibodies (CXCL8: #500-P28, IFNβ: #500-P32B; PeproTech) and detecting biotinylated rabbit polyclonal antibodies (CXCL8: #500-P28Bt, IFNβ: #500-P32BBT; PeproTech) were used. After the addition of streptavidin-horseradish peroxidase (#016-030-084; Jackson Immunoresearch Laboratories, West Grove, PA, USA), substrate TMB/E solution (#ES001; Millipore, Temecula, CA, USA) was added. For IL-6 and IFNγ, the following ELISA kits were used: IL-6 (#900-TM16; Peprotech), IFNγ (#900-TM27, Peprotech). The reaction was stopped by the addition of 0.18 M H₂SO₄ and was measured at 450 nm.

The T. cruzi recombinant protein TcCyP19 was separated by SDS-PAGE and electrotransferred from polyacrylamide gels onto nitrocellulose membranes in Tris 25 mM, glycine 192 mM and 20% v/v of methanol in Mini Protean II (Bio Rad, Hercules, CA, USA) equipment at 30 V overnight at 4 °C. Strips were blocked in 5% skimmed milk in PBS at room temperature (RT) for 1 h and then incubated at RT for 1 h with polyclonal mouse anti-TcCyP19 (1:2000). For the detection of TcCyP19 in the sera from mice and humans, strips were incubated for 1 h at RT with sera from anti-TcCyP19 elicited in mice (1:1000), an uninfected human, a chronic adult T. cruzi-infected patient, an uninfected mouse, and a chronic and acute T. cruzi-infected mouse, all diluted to 1:100. Membranes were washed with PBS-Tween20 and then incubated at RT for 1 h with biotinylated anti-mouse IgG (Jackson, West Grove, PA, USA) (1:2000) or goat anti-human Horseradish Peroxidase (Invitrogen, Waltham, MA, USA) (1:5000) (Abcam, Cambridge, United Kingdom). After washing, membranes were incubated with streptavidin-horseradish peroxidase (Jackson) (1:1000) at RT for 30 min. Detection was performed with alpha-chloronaphtol.