Goat anti rabbit fitc

Cells were harvested by trypsinization and fixed in fixation buffer (Nordic MUbio, Susteren, Netherlands). Cells were incubated with primary and secondary antibodies in permeabilization buffer (Nordic MUbio) for 20 min each and were subsequently analyzed using the FACSCanto II cytometer and the FACSDiva Software v6 (BD Biosciences). The following antibodies were used for staining: rabbit anti-p-EGFR Tyr1068 (cell signaling), rabbit IgG isotype control and goat anti-rabbit FITC (Life Technologies, Darmstadt, Germany). Median specific staining intensity (MFI) was calculated as ratio of the fluorescence intensity of cells stained with the specific antibody and the isotype control.

The immunofluorescence procedure was performed according to our previous study [4 (link)]. The primary antibodies were used as described for Western blotting, and goat anti-rabbit FITC and goat anti-mouse TRITC secondary antibodies were purchased from Thermo Fisher. ER-Tracker Red (C1041) was purchased from Beyotime Biotechnology (Shanghai, China); a cell autophagy fluorescent detection kit (G0170) was purchased from Solarbio Life Sciences (Beijing, China); and Ad-GFP-LC3 (HBAD-1006) and Ad-mRFP-GFP-LC3 (HB-AP210 0001) were purchased from HANBIO (Shanghai, China). The images were obtained by confocal laser scanning microscopy (CLSM), and the fluorescence intensity was analyzed using ImageJ software.

EAhy cells were fixed and stained for CCL5 and CXCL4 as described above. Visualization was performed using Goat-anti-Rabbit FITC (Thermo). Cells were imaged on a Leica TCS SP8 Confocal microscope with a 100× oil immersion/1.4 NA objective and 2× optical zoom, with excitation at 405 or 488 nm, and emissions were collected at 413–480 nm and 498–580 nm, respectively. Images of 512 × 512 pixels were obtained with a pixel size of 0.09 µm, standard pinhole size, and scan speed of 400 Hz. Z-stacks were made with a step size of 0.219 µm (Movie S1) or 0.3 µm (Figure S1B).

Synchronous cultures of NF54 and FCR3 mainly at schizont stage were used to prepare IFA slides. Culture was washed trice with RPMI, spinning for 5 min at 2000 rpm. After centrifugation Fetal Calf Serum (FCS - Gibco, Invitrogen, Breda, The Netherlands) was added in 1:1 ratio to the pellet. Thin smears were prepared on multitest 12-well slides (MP Biomedicals, Eindhoven, The Netherlands). Slides were stored in slide boxes placed in a plastic bag containing desiccant - silica gel (Sigma, Zwijndrecht, The Netherlands) at − 80 °C until use.
At room temperature, thawed slides were fixed with cold methanol for 10–60 s. 15 μL of day 70 obtained sera (primary antibody) at different dilutions, starting at 1:1000 in 2-fold, was added to each slide well and incubated for 1 h at RT. After five PBS (Gibco, Invitrogen, Breda, The Netherlands) washes, slides were incubated again for 1 h in a moist box with secondary antibody Goat-anti-Rabbit-FITC (Thermo Fisher Scientific, Etten-Leur, The Netherlands) diluted 100x in PBS containing 1% FCS. Then slides were washed again five times. Nuclei were stained with DAPI at 1:5000 in antifade (Sigma, Zwijdrecht, The Netherlands). As a positive control sera obtained from BG98 rabbits was used [13 (link)]. The IFA titre is expressed as an end-point titre, i.e. the highest dilution at which a positive reaction was observed.

Immunofluorescence staining was performed after 1 week of co-culture if not stated otherwise. For staining of cells within a 3D fibrin-matrix, hydrogels were fixed with 4% paraformaldehyde overnight and then washed prior to adding the primary antibody diluted in 1 × phosphate-buffered saline (PBS)/1% bovine serum albumin (BSA). Primary antibody was incubated overnight and afterwards matrices were washed followed by overnight secondary antibody incubation. All steps were performed at 4 ° C on a shaker. Staining was performed against collagen type IV (rabbit-polyclonal, Abcam, Cambridge, MA, UK), laminin (rabbit-polyclonal, Abcam, Cambridge, MA, UK) or E-Selectin (BD Pharmingen, San Diego, CA, USA). Secondary antibodies goat anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific Inc., Waltham, MA, USA) or goat anti-rabbit-FITC (Thermo Fisher Scientific Inc., Waltham, MA, USA) were used. For immunofluorescence staining in a two-dimensional (2D) setup, cells were fixed with 4% paraformaldehyde for 20 min. Anti-CD31-FITC (BD Pharmingen, San Diego, CA, USA) was diluted in 1 × PBS/1% BSA and incubated for 45 min. All images were taken on an epifluorescence microscope (Zeiss Observer A1, Zeiss, Oberkochen, Germany) or on a laser-scanning confocal microscope (Zeiss LSM510, Zeiss, Oberkochen, Germany).