Donkey anti goat af488

Five candidate genes were validated using two or three sgRNAs to the genes. A sgRNA that does not target the mouse or human genomes was included as a negative control. The sequences of the guides are listed in Supplementary Table 2. The sgRNAs were cloned into lentiCRISPR v.2 (Addgene, 52961) and packaged in HEK 293T cells with psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 2259) using Lipofectamine 3000 (Thermo Fisher). ΔB4galt7 N2a cells²³ were transduced and selected for 7 days in the presence of puromycin. Clonal LDLR-deficient cells were obtained by limiting dilution. Ldlr gene editing was validated by next-generation sequencing on an Illumina HiSeq 2500 platform (Genome Technology Access Center of Washington University) with 300-base-pair paired-end sequencing. Ldlr gene editing also was confirmed by flow cytometry analysis of LDLR surface expression using a goat-anti-mouse LDLR antibody (Thermo Fisher, PA5-46987, 1:100 dilution) and detection with a donkey-anti-goat-AF488 (Thermo Fisher, A-11055, at dilution of 1:1,000). When staining THP-1 cells, a 1:200 dilution of Fc block (Biolegend, 422301) was added according to the manufacturer’s instructions.

Coronal sections obtained from mice that underwent either standard experimental procedure for transcardial perfusion assessment as described above, or the one without TRITC-dextran injection were fixed with 4% methanol-free formaldehyde (Thermo Fisher Scientific, #28908) for 1 h. After three PBS washing steps (5 min each), the sections were blocked with a solution consisting of 5% donkey serum (Millipore, #S30-100ML) and 0.3% Triton X-100 in PBS for 3.5 h. After blocking, the samples were incubated with primary antibodies—goat anti-type IV collagen (1:500; SouthernBiotech, #1340-01) overnight at 4 °C. After three PBS washing steps, the sections were incubated with secondary antibodies—donkey anti-goat AF488 (1:500; Thermo Fisher Scientific, #A-11055) and Hoechst (Thermo Fisher Scientific, #H3570) for 4.5 h at RT. Afterward, the sections were washed and mounted with ProLong™ Diamond Antifade Mountant mounting media (Thermo Fisher Scientific, #P36970). Mounted sections were imaged with slide scanner as described above. High-magnification images were obtained using a confocal microscope using 63×, NA 1.4 objective (Zeiss Microscopy, LSM 780).

Following 6 hours of co-culture, spheroids were washed, fixed with 4% paraformaldehyde (ThermoScientific), permeabilized in PBS 0,5% Triton-X100 and blocked in PBS 1% BSA (Sigma Aldrich). Primary anti-human CD8a (Biolegend) and anti-human CD4 (R&D Systems) antibodies were added and incubated before DNAHoechst (Invitrogen), secondary rat-anti-mouse AF647 (Biolegend) and donkey-anti-goat AF488 (Thermo Scientific) antibodies were added and incubated for 24 h. For mesothelin staining, spheroids were incubated with PE-conjugated anti-human MSLN antibody (R&D Systems) for 24 h. All stainings were performed in PBS 1% BSA at 4°C. Spheroids were cleared overnight in PBS 88% glycerol (Sigma Aldrich), transferred to ibidi 8-well plates and imaged at the LSM900-airy confocal microscopy. At least three spheroids were imaged per condition, with three images at 10 µm distance per spheroid. Quantification was performed in QuPath.^{25 (link)}

Immunofluorescence staining was performed by iHisto Inc. (ihisto.io) Samples were processed, embedded in paraffin, and sectioned at 4 μm. Paraffin sections were then deparaffinized and hydrated using the following steps: 15 min in xylene twice; 5, 5, and 5 min in 100%, 100%, and 75% ethanol, respectively; and 5 min in PBS at room temperature repeated three times. Heat-induced epitope retrieval (HIER) was achieved by pressure cooker (SB Bio) for 15 min with sodium citrate buffer and 20 min of cooling down at room temperature. Sections were then washed with PBS three times, and blocked with 3% bovine serum albumin for 30 min. The sections were incubated with the primary antibody Anti-IL6 (GB11117, 1:100) and anti-VEGF (R&D systems AF293, 1–100) overnight at 4 °C. Sections were rinsed with PBS and incubated with Donkey anti-Goat AF488 (Invitrogen, A32814, 1:500) for 1 h at room temperature followed by 3 times of wash. Subsequently, the sections were with incubate with Goat anti-rabbit AF647 (Invitrogen, A21245, 1:500) and Goat anti-mouse AF555(Invitrogen, A21424, 1:500) for another 1 h. Lastly, quench autofluorescence with Sudan Black and stain DAPI. Whole slide scanning (40×) was performed on a Panoramic midi scanner (3D histech).

BCA sections were deparaffinized and rehydrated in xylene and ethanol series. After antigen retrieval (H-3300-250, Vector Laboratories), sections were blocked with fish skin gelatin–PBS (6 g/L) containing 10% horse serum for 1 hour at room temperature. Slides were incubated with the following antibodies: mouse monoclonal α-SMA–Cy3 (4.4 μg/mL, clone 1A4, C6198, Sigma-Aldrich), goat polyclonal anti-GFP (4 μg/mL, ab6673, Abcam) for detection of eYFP, and rabbit polyclonal p21 (18 μg/mL, 10355-1-AP, Proteintech). TUNEL (30074, Bioutium) was used for staining. The secondary antibodies donkey anti-goat AF-488 (Invitrogen, A11055) conjugated to Alexa 488 (5 μg/mL) and donkey anti-rabbit conjugated to Alexa 647 (A31573, 5 μg/mL) were used (Thermo Fisher Scientific), and DAPI (0.05 mg/mL, D3571) was used as a nuclear counterstain; slides were mounted using Prolong Gold Antifade (P36930), purchased from Thermo Fisher Scientific.

Adherent cultures and hydrogels were fixed with 4% paraformaldehyde at room temperature, then permeabilized using 0.1% TritonX-100 and incubated with blocking solution (PBST with 10% goat serum and 3% BSA) for 1 h at room temperature and stained with the appropriate primary antibody in blocking buffer for 1–2 h at room temperature or overnight at 4 °C. The samples were then washed with PBS, incubated with a secondary antibody, washed and stained with DAPI or Hoechst 34580. Primary antibodies used in this study were: DDR1 (5583, Cell Signaling Technology, clone D1G6, 1:100), E-Cadherin (ab1416, Abcam, Clone HECD-1, 1:100), JAG1 (PA5-46970, Invitrogen, 1:100), Notch1 (4380, Cell Signaling Technology, clone D6F11, 1:200), CK18 (4548 Cell Signaling Technology, clone DC10, 1:250), p63 (13109, Cell Signaling Technology, clone D2K8X, 1:250), CK14 (RB-9020-P, Thermo Fisher, 1:200). Secondary antibodies used were: Rat anti-mouse-APC (550874, BD Bioscience, 1:500), Donkey anti-rabbit-AF555 (A-3157, Invitrogen, 1:500), Donkey anti-rabbit-AF555 (A-31572, Invitrogen, 1:500), Donkey anti-goat-AF488 (A-11055, Invitrogen, 1:500). Donkey-anti-mouse-AF555 (A-31570, Invitrogen, 1:500). Dyes and probes used for immunofluorescence: Phalloidin-AF647 (A22287, Life Technologies, 1:400 of 400× stock), DAPI (D1306, Life Technologies, 1 ug/ml), Hoechst 34580 (H21486, Life Technologies, 1 µg/ml).

Formalin-fixed and paraffin-embedded samples from 22 primary human BL cases and from tonsils were obtained from the histopathology files of the Pathology Institute, University Wuerzburg. When biopsy material was studied, the permission and consent of patients was received before. Immune stains of paraffin slices were performed with Abs directed against NFATc1 (clone 7A6, BD Pharmingen) and NFATc1/α (anti-IG-457). All pictures were captured with an Olympus Color view camera mounted on an Olympus BX41 dual-head light microscope. The pictures were taken with a Leica Confocal Laser Scanning Microscope (TCS SP5 II) and were analyzed with the Leica Software Image Pro Plus. For further demonstration, the digital images were processed using Adobe Photoshop CS3, Irfan view or Microsoft Office Power Point 2010.
For confocal microscopy, Namalwa and Ramos cells, Eµ-MYC induced mouse primary tumor cells,(#1542T), B cell lymphoma (BCL) cells from the same tumor (#1542B) and naïve splenic B cells were incubated for 1 h with αNFATc1 (7A6), αNFATc1/α (anti-IG-457) and, in some assays, αKi-67. Next, they were stained by secondary fluorescent labelled goat-anti-mouse Alexa Fluor (AF) 488, or goat-anti-rabbit AF 555, or AF 488, or by strepavidin AF 488 (all from eBiosciences), or goat-anti-mouse AF 647 (Dianova), or donkey anti-goat AF 488 (Invitrogen).