Alexa fluor 594

Double immunofluorescence was performed based on a previously reported technique for double immunohistochemistry [49 (link)] and immunofluorescence [50 (link)] with modifications. Briefly, a two-color immunofluorescence stain was performed using P-glycoprotein (green color) and AR (red color) antibodies. Tissue samples were deparaffinized, and antigen retrieval was performed using citrate buffer solution (pH 6.) in a commercial pressure chamber (Dako, Carpinteria, CA, USA) for 30 s at 125 °C, followed by 25 min at 94 °C, and cooling for 15 min. Then, unpacific binding proteins were blocked using a commercial solution for 30 min (Dako, Carpinteria, CA, USA). Anti-rabbit polyclonal AR (1:50) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 484 (Invitrogen, Carlsbad, CA, USA) at a 2 μg/mL dilution in phosphate-buffered saline (PBS) for 1 h and the slides were washed using PBS solution. The second primary antibody was applied (1:700 monoclonal mouse anti-P-gp clone C494, BioLegend, San Diego, CA, USA) overnight. Then, the secondary goat anti-mouse antibody was conjugated with Alexa Fluor^® 594 (BioLegend, San Diego, CA, USA), for one hour. Slides were counterstained with 4-6-diamidino-2-phenylindole (DAPI; Sigma, Portland, OR, USA) and evaluated using a laser scanning confocal microscope (Leica Biosystems, Wetzlar, Germany).

Air-dried cryostat sections (4 μm thickness) were fixed (methanol, 8 min). Unspecific binding sites were blocked in 2% BSA (Roth, Karlsruhe, Germany) for 1 h followed by incubation with 1 μg of the following AlexaFluor488, AlexaFluor594, and AlexaFluor 647-labeled mAbs: CD4, CD8α, CD11b, Gr1, CD11c, F4/80, CD104, CD206, PD-1, and PD-L1 (all from Biolegend). For intracellular stainings, slides were fixed in 4% paraformaldehyde w/o methanol (Thermo Scientific, Darmstadt, Germany, 30min) and cells permeabilized in 0.5% Triton X−100 (Sigma-Aldrich, Darmstadt, Germany, 15 min). After blocking with 2% BSA (Serva, Heidelberg, Germany), slides were incubated with the monoclonal rabbit anti-IRF5 antibody (1:50; ThermoFisher Scientific, Darmstadt, Germany) overnight at 4 °C, followed by a secondary goat anti-rabbit Alexa647 antibody (1:500; Cell Signaling, Frankfurt am Main, Germany). Sections were washed, embedded in Roti Mount Flour Care DAPI (Roth), and target proteins visualized on a confocal laser scanning microscope (Elyra 7, Zeiss, Jena, Germany) using 20× objectives. IRF5⁺ cells were quantified by counting individual positive cells in three high power fields per sample (n = 3/group).

Cells were fixed with 2% paraformaldehyde (PFA) w/o methanol (15 min, Thermo Fisher Scientific, Darmstadt, Germany), permeabilized, and blocked with 0.5% Triton X-100 (Thermo Fisher Scientific, Waltham, USA) in 2% BSA (PAN-Biotech, Aidenbach, Germany) for 60 min. ER stress markers included: Alexa 647 anti-ATF-4 antibody (B-3, 1:50, Santa Cruz), Alexa 594 anti-calnexin antibody (AF18, 1:50, Santa Cruz), and Alexa 488 anti-cytochrome c (1:50, Biolegend). For stemness, antibody mixtures, either containing anti-GFAP (1:200, Alexa Fluor 594, BioLegend, San Diego, USA) and anti-A2B5 (1:200, Alexa Flour 647, BioLegend, San Diego, USA), or anti-Oct-4 (1:500, Alexa Fluor 647, BioLegend, San Diego, USA) and anti-Nanog (1:500, Alexa Flour 488, BioLegend, San Diego, USA) were added and staining was done at 4 °C overnight. The next day, GFAP/A2B5-antibody mix was stained with Phalloidin green (1:50, BioLegend, San Diego, USA). Nuclei were counterstained with DAPI (1:1.000, Biomol, Hamburg, Germany) and cells were analyzed using a Zeiss microscope Axio Observer 7 (Zeiss, Oberkochen, Germany).

Tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series. After heat-induced epitope retrieval in citrate buffer, pH 6.0 (Scytek Laboratories, Inc. West Logan, UT, USA) for 40 min, sections were incubated with 3% bovine serum albumin blocking reagent for 10 min at room temperature. Subsequently, tissue sections were incubated with anti-vascular endothelial growth factor A (VEGFA) (Abcam, Cambridge, UK), anti-Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1, NOVUS, Briarwood, CO, USA), and anti-human Nuclear (Biorbyt, Cambridge, UK) primary antibodies (1:100) for 90 min at room temperature, followed by incubation with Alexa Fluor 488 (Biolegend, San Diego, CA, USA) and Alexa Fluor 594 (Biolegend, San Diego, CA, USA) secondary antibody (1:500). The sections were mounted with DAPI-containing mounting medium (Vector Laboratories Inc., Burlingame, CA, USA) and observed under an inverted microscope (Zeiss MicroImaging GmbH, Jena, Germany). The proportion of VEGFA-positive cells was measured using Image J software (version 1.53K; National Institutes of Health, Bethesda, MD, USA) in five microscopic fields in each stained sample. LYVE-1 and hNuclear-positive cells were counted manually in five microscopic fields in each stained sample, and the mean value was used for statistical analyses.

The HA epitope was detected with mouse monoclonal antibody (MAb) HA.11 (BioLegend; 901515), rabbit polyclonal antibody (pAb) anti-HA (Invitrogen; PI715500), or mouse MAb HA.11 conjugated to Alexa Fluor-594 (BioLegend 901511). The Ty1 epitope was detected with mouse MAb BB2 (58 (link)). The c-Myc epitope was detected with mouse MAb 9E10 (59 (link)). The V5 epitope was detected with mouse MAb anti-V5 (Invitrogen; R96025). The smOLLAS epitope was detected with rat MAb anti-OLLAS (60 (link)). Toxoplasma-specific antibodies include pAb rabbit anti-IMC6 (49 (link)), mouse MAb anti-ISP1 (23 (link)), pAb rabbit anti-IMC3 (61 (link)), pAb rabbit anti-IMC12 (41 (link)), pAb rabbit anti-ROP13 (62 (link)), MAb mouse anti-F₁β subunit (5F4) (39 (link)), MAb mouse anti-ATrx1 (11G8) (63 (link)), pAb guinea pig anti-NHE3 (64 (link)), MAb mouse anti-MIC2 (65 (link)), MAb mouse anti-ROP7 (66 (link)).

Spleens were harvested, fixed with 4% paraformaldehyde, washed in PBS, and dehydrated overnight in 30% sucrose (Sigma). The samples were embedded in Tissue-TekOCT compound (Bio-Optica) and frozen in an ethanol dry ice bath. Sections of 7 μm were placed on glass slides (Bio-Optica) and blocked for 30 min with PBS-Tween 0.05% plus 0.5% FBS. Sections were stained with Abs directed against B220 (Alexa Fluor 594, Biolegend, 103254, San Diego, CA, USA) for 1 h at 37 °C to visualize B cells, nuclei were counterstained using DAPI (KGA215, Jetway, Nanjing, China) for another 15 min at 37 °C, then coverslips were mounted on a microscope slide with Antifade Mounting Medium (P0126, Beyotime, Jiangsu, China). Finally, Images were acquired using a laser scanning confocal microscopy (LSM880, Zeiss, Jena, Germany).