Alexa 633 goat anti mouse igg

The following primary antibodies were used: mouse anti-GFP (Covance MMS-118P; RRID:AB_291290; WB: 1:3000), mouse anti-HA-7 (Merck H9658; RRID:AB_260092; WB 1:20,000) mouse anti-T7-tag (Merck 69522; RRID:AB_11211744; WB 1:10,000) and mouse anti-PSD-95 (Thermo Fisher Scientific MA1-046, RRID:AB_2092361; ICC: 1:500); rat anti-RFP (Chromotek 5F8; RRID:AB_2336064; WB: 1:1000); chicken anti-MAP2 (Antibodies-Online; RRID:AB_10786841; ICC: 1:1000). HRP-labeled goat secondary antibodies were from Jackson ImmunoResearch (RRID:AB_2338133; RRID:AB_10015289) and used for WB at 1:2500 dilution. For ICC, Alexa 633 goat anti-mouse IgG (Invitrogen A21050; RRID:AB_2535718) and Alexa 405 goat anti-chk IgG (abcam ab175675; RRID:AB_2819980) were used at 1:1000 dilution.

The following primary antibodies were used: mouse anti-GFP (Covance MMS-118P-500, RRID:AB_291290; WB: 1:3000); rat anti-RFP (Chromotek 5F8ChromoTek 5f8-100, WB: 1:1000); chicken anti-MAP2 (antibodies Antibodies-Online ABIN361345, ICC: 1:1000); mouse anti PSD-95 (Thermo Fisher MA1-046; ICC: 1:500); rabbit anti-HA (Sigma Aldrich #H9658 ICC 1:200); rabbit anti-CaMKIIα (abcam ab52476); and rabbit anti-CaMKIIα phospho-T286 (abcam ab32678). HRP-labeled goat secondary antibodies were purchased from Jackson ImmunoResearch and used for WB at 1:2500 dilution. For ICC, Alexa 633 goat anti-chk IgG (Invitrogen A21103), Alexa 633 goat anti-mouse IgG (Invitrogen A21050), Cy3 goat anti-rabbit IgG (abcam ab6939), and Alexa 405 goat anti-chk IgG (abcam ab175675) were used at 1:1000 dilution.

Previously fixed coverslips were subjected to two washes with 1× PBS (5 min each). Blocking was performed for 30 min using 1 % bovine serum albumin (BSA) in 1× PBS at RT. Primary antibodies used were as follows: Mouse anti-Lamin B2 (Abcam (ab8983), 1:600) and Rabbit anti-Lamin A (Abcam (ab26300), 1:500). Antibody dilutions were prepared in 0.5 % BSA in 1× PBS and incubated for 90 min at RT. Secondary antibodies used were goat anti-mouse IgG–Alexa 633 (Invitrogen (A-21052) 1:1000) and goat anti-rabbit IgG–Alexa 488 (Invitrogen (A-11034) 1:1000) for 1 h at RT. Post fixation and post permeabilization were subsequently performed with 4 % PFA (5 min RT) and 0.5 % Triton X-100 in 1× PBS (5 min RT), respectively. This was followed by two washes each with 1× PBS (5 min each) and 50 % FA/2× SSC (pH 7.4). The probe for ZNF570 was equilibrated at 37 °C for 5 min followed by denaturation at 80 °C for 5 min and quick chilled on ice for 2 min. Pre-annealing was performed at 37 °C for 30–40 min. This denatured probe (3–4 μL) was spotted onto the fixed cells that were subjected to immunostaining for Lamin A and Lamin B2 and subjected to co-denaturation at 80 °C for 5 min. Hybridization was for 48 h in a humidified box at 37 °C.

As described above, rhFLT3-L and IL-6-activated BM cells were transferred to polylysine-coated glass wells with 10 ng/ml rmGM-CSF, 10 ng/ml rmIL-4. After 24 hrs culture, the DCs were pulsed with the peptides and stimulated with LPS. The cells were washed, fixed for 2 min in ethanol (-20°C) and blocked overnight with 1% BSA (Sigma) in PBS, washed and stained for 1hr with anti-MUC1 (BC2-Alexa488) and anti H-2K^b (AF6-88.5, BD Pharmingen), followed by a secondary goat anti-mouse IgGAlexa633 (Invitrogen) at 1:100 dilution for 20 min at room temperature, washed and mounted with Prolong Gold Antifade Reagent (Invitrogen). Imaging was performed on a Zeiss laser scanning microscope with a 63X 1.4 NA oil DIC immersion objective and analyzed using Zeiss LSM image browser.

The following primary antibodies were used for immunofluorescence microscopy: human anti-centromere (CREST serum 1:3, Antibodies Inc., 15-234-001; Antibodies Inc., Davis, CA, USA), mouse monoclonal anti-lamin A/C (1:30; Abcam, ab40567; Abcam, Cambridge, UK), monoclonal anti-LAP2α (1:10; clone 15/2, kind gift of Dr. Roland Foisner, Max F. Perutz Laboratories, Vienna, Austria), monoclonal antibody mAb414 (1:2000; Covance, MMS-120R; Covance, Emeryville, CA, USA), monoclonal anti-Hec1 (1:200; clone 3G9, Abcam, ab3613), and rabbit polyclonal anti-LAP2α (1:1000; Abcam, ab5162), polyclonal anti-lamin A (1:500; Sigma-Aldrich, L1293; Sigma-Aldrich, Diegem, Belgium), polyclonal lamin B1 (Abcam, ab16048), polyclonal anti-Sun1 (1:1000, kind gift of Dr. Ulrike Kutay, ETH Zurich, Switzerland), polyclonal anti-Sun2 (1:100; Sigma-Aldrich, HPA001209), polyclonal anti-emerin (1:1000; Bethyl Laboratories, A304-491A; ImTec Diagnostics, Antwerpen, Belgium), as well as polyclonal anti-Nesprin-2 (1:50, kind gift of Dr. Iakowos Karakesisoglou, Durham University, UK). Secondary antibodies were the corresponding goat anti-mouse IgG Alexa 568 (1:1000; Invitrogen), goat anti-mouse IgG Alexa 555 (1:1000; Invitrogen), goat anti-rabbit IgG Alexa 568 (1:1000; Invitrogen), goat anti-mouse IgG Alexa 633 (1:350; Invitrogen), and goat anti-rabbit IgG Alexa 633 (1:350; Invitrogen).

Exosomes isolated from cancer cells were labelled with PKH67 dye (Cat. #: P7333; Sigma Aldrich, Saint-Louis, USA) according to the manufacturer's instructions. Then, fluorescently-labeled exosomes were incubated with endothelial cells (HUVEC, seeded at 30,000 cells in 24-well plate) for 2-4 h. Following this treatment, the cells were fixed for 10 minutes in 4% PFA, washed two times with PBS and blocked for 30 min with PBS/BSA (2%). In order to visualize the plasma membrane, HUVEC were labelled with anti-CD31 (dilution 1:20; Cat. N#: M0823, Dako) for 2h at RT. Following three PBS washes, the samples were incubated with Alexa633 goat-anti-mouse IgG (dilution 1:2000; Cat. #: A21050, Life Technologies) for 45 min. After PBS wash, the slides were mounted (Fluorescent Mounting Medium; Cat. #: S3023, Dako) and visualized under a confocal microscope. Images were acquired using TCS-SP5-A0BS confocal microscope (Leica, Wetzlar, Germany) at a magnification of 63X.
Exosome uptake was quantified using the particle analyzer of Fiji/ImageJ 1.51d [51 (link)] after image binarization. Colocalization of uptaken PKH67-labelled exosomes and CD31 was evaluated after confocal Z-stack image acquisition. Split channel images were submitted to a Pearson's correlation analysis using the Fiji/ImageJ 1.51d Coloc 2 plugin.