Alexa fluor 647 goat anti mouse antibody

Cells were stained to detect CaSR expression using a monoclonal antibody against the ADD region in the N-terminal domain of the CaSR sequence. Cells grown on glass cover slips were fixed in 3.7% paraformaldehyde for 20 min, permeabilized with 0.2% Triton-X for 20 min, and blocked with 5% goat serum for 30 min. Cells were incubated with anti-CaSR antibody (1:200, Abcam, UK) for 1 h at room temperature. After extensive washing, samples were incubated with Alexa Fluor 647 goat-anti-mouse antibody (1:1000, Life Technologies). Nuclei were stained with DAPI (1:3000, Roche, Switzerland). Whole slide images were acquired using TissueFAXS (TissueGnostics, Austria). Isotype-specific IgG was used as negative control and human kidney tissue was stained as positive control.

Cells (MCF7/TGL/pQ, MCF7/TGL/HA‐Bcl‐xL, MCF7/TGL/HA‐Bcl‐xL^R132W, MCF7/TGL/HA‐Bcl‐xL^{N136K (Clone 5)}, MCF7/TGL/HA‐Bcl‐xL^R165W, and MCF7/TGL/HA‐Bcl‐xL^A201T) were cultured on glass coverslips for 24 h before fixation in 4% paraformaldehyde in PBS for 10 min. After permeabilizing and blocking in 0.5% BSA in PBS with 0.025% Triton X‐100, 0.02% NaN₃, and 0.3 mM DAPI for 2 h, coverslips were incubated with primary antibody (mouse anti‐HA 1:200; Cell Signaling Technology, 2367) overnight at 4°C. The coverslips were then washed four times with PBS for 5 min each, followed by incubation with Alexa Fluor 647 goat antimouse antibody (1:400; Life Technologies, A21236) at room temperature in the dark for 2 h, and were washed four times with PBS for 5 min each. Coverslips were mounted with VectaShield mounting medium (Vector Labs) and cells were examined using confocal microscopy (Olympus FLUOVIEW FV10i). The total, nuclear, and cytoplasmic immunofluorescence signals of more than 40 cells per cell line from a minimum of 10 separate images were quantified using Fiji ImageJ (version 1.51p).

A cell-based ELISA as described previously²⁶ was used to determine the anti-spike glycoprotein antibody response in the mouse sera and convalescent plasma. Briefly, MDCK-Spike was produced by stably transfecting parental MDCK-SIAT1 cells with full-length SARS-CoV-2 spike glycoprotein cDNA using a lentiviral vector. MDCK-Spike cells (3 × 10⁴ cells/well) were seeded in 96-well plates and incubated overnight at 37 °C. Mouse sera was diluted as above and 50 μL was transferred to the washed plates seeded with MDCK-Spike cells for 1 h at RT. Human plasma was pre-incubated with MDCK-SIAT1 cells as described above, before dilution and adding to MDCK-Spike for 1 h at RT. Parallel plates were seeded with MDCK-SIAT1 for background subtraction as described above for human plasma. For mouse sera, 50 μL of a secondary Alexa Fluor 647 goat-anti-mouse antibody (1:500) (Life Technologies A21235) was then added for 1 h at RT. For human sera, 50 μL of a secondary Alexa Fluor 647 goat-anti human antibody (1:500) (Life Technologies A21455) was used. Plates were then washed with PBS and 100 μL of PBS/1% formalin was added to each well. Fluorescence signal was read on a Clariostar plate reader and the EPT titre was calculated as described above.

Neutrophils (1 × 10⁶) were seeded on a sterile round glass cover slip that was placed in a 24-well cell culture plate. As described above in the flow cytometric assay for NETs, 100 μM PMA or 1 μg/ml VP1 was added. After 4 h of incubation, the glass cover slips with the attached cells were carefully removed from a 24-well culture plate and fixed with ice-cold acetone. Then, samples were blocked with 5% goat sera and stained overnight with a rabbit polyclonal antibody to histone 3 (citrulline R2 + R8 + R17) (1:300, ab5103, Abcam) and with a mouse anti-MPO antibody (1:300, ab90810, Abcam). The samples were washed in PBST and further stained with an Alexa Fluor® 555 goat anti-rabbit antibody (1:500, Life Technologies, USA) and an Alexa Fluor® 647 goat anti-mouse antibody (1:1000, Life Technologies, USA). Nuclei in the samples were stained with 4′6-diamidino-2-phenylindole. Images were captured by Zeiss LSM7 confocal fluorescence microscopes using the appropriate lenses and filters.
For experiments in vivo, the left lower lung of mice that were or were not treated with VP1 was carefully harvested, frozen in Tissue-Tek OCT media and prepared into 7-μm-thick slices. The fixation, blocking, antibody incubation, and image acquisition of these sections were performed as described at the cell level.