Prolong gold antifade mountant containing 4 6 diamidino 2 phenylindole dapi

Cells were seeded (3 mL, 1.2 × 10⁶ cells) in six-well plates containing sterile coverslips and incubated under standard CHO culture conditions to approximately 60% confluence. PBS-washed cells were fixed with 4% paraformaldehyde. Cells were washed three times with a modified TBS buffer, mTBS (20 mM Tris–HCl, pH 7.2, 100 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂), blocked with 1% bovine serum albumin (BSA, ≥99%, globulin-free)(Sigma-Aldrich Merck, Gillingham, UK) for 30 min then washed as before with mTBS. Fluorescein isothiocyanate (FITC)-labelled lectins (EY Laboratories, San Mateo, CA, USA) SNA-I and MAA (containing both MAA-1 and MAA-2) were prepared in mTBS (40 μg/mL), added to coverslips (0.5 mL, 20 μg), and incubated for 2 h at 4 °C in the dark in the absence or presence of 1 mL 100 mM lactose. Plates were washed three times with mTBS and then with PBS. The coverslip with the attached cells was mounted on a glass slide with one drop of ProLong Gold antifade mountant containing 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA, USA) and allowed to dry overnight in the dark. Images were acquired using an upright microscope (Olympus BX53) with violet (autofluorescence/DAPI) and green (FITC) filters.

Air-dried thin blood smears were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, followed by permeabilization with 0.1% Triton X-100 in PBS. Blocking and antibody binding steps were performed in PBS containing 3% bovine serum albumin. Dual staining experiments were performed sequentially, starting with rat anti-HA, to eliminate cross-reactivity of the anti-rat secondary antibody with mouse or rabbit IgG. The secondary antibodies used were anti-rat IgG antibody conjugated to Alexa Fluor 488, anti-rabbit IgG conjugated to Alexa Fluor 594, and anti-mouse IgG conjugated to Alexa Fluor 594, all highly cross-adsorbed. Slides were mounted in ProLong Gold Antifade Mountant containing 4′,6′-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Images were acquired at ×100 magnification using a Nikon Eclipse Ti fluorescence microscope fitted with a Hamamatsu C11440 digital camera and overlaid in ICY bioimage analysis software (icy.bioimageanalysis.org).