4.2.10. Immunofluorescence Staining of Heparin and Heparin-Sulfate Surfaces

For immunofluorescence, APTES coated coverslips were incubated with streptavidin (200 nM) for 10 min prior to a fixation step (2.5% glutaraldehyde, 2.0% paraformaldehyde in PBS) for another 15 min. Samples were washed three times for 2 min with PBS (3 × 2:00). Surfaces were then incubated with biotin–heparin and biotin–heparan sulfate (800 nM) in PBS for 30 min. Samples were washed again with PBS (3 × 2:00). Samples were then blocked with bovine serum albumin (4% in PBS) for another 30 min. After blocking, samples were washed three times using the kinetic buffer (10 mM HEPES, 10 mM NaCl, 0.05% Tween 20, pH 7.4). Spike protein (50 nM) was incubated with Mouse Anti 6x-His Tag Antibody conjugated with Alexa Fluor 488 (1 μg/mL) for 1 h. Surfaces were washed with kinetic buffer (3 × 2:00) and were then incubated with mixture of spike-Ab for 1 h. Samples were then incubated with NTD Ab (1 μg/mL) in lateral flow assay buffer for 1 h. Finally, surfaces were incubated with Antihuman Alexa 594 in PBS for 1 h. Samples were washed with PBS and mounted onto glass slides using Immu-Mount. Samples were then imaged on a Ziess 710 confocal microscope.

Data analysis was done using FIJI. Prior to processing, immunofluorescence images were first blurred using a Gaussian threshold (diameter: 2 pixels) and a rolling pin filter for background subtraction (50 pixels). Protein locations were then identified through an automatic threshold using either a max entropy or triangle algorithm. Single randomly bright pixels were then removed using the “analyze particles” function to remove particles smaller than 0.5 μm². Proteins with both NTD Ab and His-Tag Ab binding were then found using the “AND” function in the “image calculator” function of FIJI. Particles were then analyzed on all three channels (NTD, His-Tag, and Combined) to determine the percentage of particles displaying both NTD and His-Tag signals.