Plan apochromat 63x 1.4 oil dic m27 objective

Overnight cultures of COL and COL type IV were back-diluted 1:200 in 20 ml TSB and grown to an OD_600 nm of 0.2, before 5 ml aliquots were transferred to test tubes. Cultures were grown for a further 1 hr, agitated at 37 °C, in the presence or absence of oxacillin (256 µg/ml for COL; 512 µg/ml for COL type IV) and 1 ml of culture was then pelleted, washed in 1 ml PBS and incubated at 37 °C agitated for 5 min with 1 µg/ml Hoechst 33342 (Invitrogen), 10 µg/ml Nile Red (Invitrogen) and 0.8 µg/ml BODIPY FL conjugated vancomycin (Van-FL, Molecular Probes). Cells were then pelleted and washed with PBS before being mounted on a 1.2% PBS agarose pad and SIM imaging was performed, due to its improved resolution compared to conventional microscopy, using a Plan-Apochromat 63x/1.4 oil DIC M27 objective, in an Elyra PS.1 microscope (Zeiss) with a Pco.edge 5.5 camera. Images were acquired using five grid rotations, with grating periods of 34 µm period for 561 nm laser (100 mW), 28 µm period for 488 nm laser (100 mW) and 23 µm period for 405 nm laser (50 mW) and images were reconstructed using ZEN software (black edition, 2012, version 8.1.0.484) based on a structured illumination algorithm, using synthetic, channel specific optical transfer functions, as described previously^{27 (link)}.

Two fluorescent antibodies, CD62P (platelet surface P-selectin) and PAC-1 (activated GP IIb/IIIa), were added to WB to study platelet activation [43 (link)]. CD62P is found on the membrane of platelets and the move to the surface of the platelet membrane. This translocation happens after platelet P-selectin is released from the cellular granules during platelet activation. The antibody PAC-1 detects the neoepitope of active GPIIb/IIIa. PAC-1 antibody binding is correlated with platelet activation.
We added 4 µL of PAC-1 (FITC-conjugated) (340507, BD Biosciences, San Jose, CA, USA) and 4 µL CD62P (PE-conjugated) (IM1759U, Beckman Coulter, Brea, CA, USA) to 20-µL WB and incubated the samples for 30 min (protected from light) at room temperature. A 10-µL drop of blood was then placed on a microscope slide, and a coverslip was then placed on the drop of blood. Following preparation, the samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany). For the PAC-1 marker, the excitation wavelength was set at 450 to 488 nm and the emission at 499 to 529 nm, while the excitation for the CD62P was 540 to 570 nm and the emission 577 to 607 nm. Unstained samples were also prepared with COVID-19 WB to assess any interference from autofluorescence.

Haematocrit samples of a few of our patients in the cohort were exposed to the two fluorescent markers, CD62P (PE-conjugated) (platelet surface P-selectin) (IM1759U, Beckman Coulter, Brea, CA, USA) and PAC-1 (FITC-conjugated) (340507, BD Biosciences, San Jose, CA, USA) (17). CD62P is a marker for P-selectin that is either found on the membrane of platelets or inside them. PAC-1 identifies platelets through marking the glycoprotein IIb/IIIa (gpIIb/IIIa) on the platelet membrane. To study platelet pathology, 4 µL CD62P and 4 µL PAC-1 was added to 20 µL haematocrit, followed by incubation for 30 min and protected from light at room temperature. The excitation wavelength band for PAC-1 was set at 450 to 488 nm and the emission at 499 to 529 nm and for the CD62P marker it was 540 to 570 nm and the emission 577 to 607 nm [17 (link)]. Samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).

Fluorescence microscopy of microclot formation in PPP was performed on some of our samples as described in our previous papers [17 (link)]. PPP were exposed to the fluorescent amyloid dye, Thioflavin T (ThT) (final concentration: 0.005 mM) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min (protected from light) at room temperature [18 (link), 27 (link)–30 (link)]. After incubation, 3 µL stained PPP was placed on a glass slide and covered with a coverslip. The excitation wavelength band for ThT was set at 450 to 488 nm and the emission at 499 to 529 nm and processed samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany).

Microclot formation in PPP samples from all 80 Long COVID/PASC patients were analysed. These patients were diagnosed by our clinical collaborators, and they were not yet placed on any clinician-initiated treatment regimens. PPP were exposed to the fluorescent amyloid dye, Thioflavin T (ThT) (final concentration: 0,005mM) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min (protected from light) at room temperature [20 (link)–23 (link)]. After incubation, 3 µL PPP was placed on a glass slide and covered with a coverslip. The excitation wavelength band for ThT was set at 450 nm to 488 nm and the emission at 499 nm to 529 nm and processed samples were viewed using a Zeiss Axio Observer 7 fluorescent microscope with a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany) [12 (link), 13 (link), 25 (link)].

Cells were prepared for imaging by attachment to coverslips coated with 0.01% Poly-L-Lysine and fixed for 15 min with 4% PFA. Cells were incubated with anti-γH2AX primary antibody (Cell Signaling Technology 9178) for 1 h at room temperature. Cells were then washed with PBS and incubated with secondary antibody for 30 min at room temperature. Cells were washed with PBS and mounted onto slides using Vectashield with DAPI. Images were acquired at room temperature using a Zeiss AXIO Observer.Z1 inverted microscope equipped with a Zeiss LSM‐880 confocal system using a Plan-Apochromat 63x/1.4 Oil DIC M27 objective. Images were then processed using OMERO (v5.4.9).

In order to determine the extent of protein deamination in fibrin networks, PPP aliquots of RA samples (n=10) and control samples (n=10) were thawed and fibrin clots prepared (refer to SEM method) on glass microscope slides in a dark room. Samples were fixed with 4% FA, washed 3× with PBS, and blocked with 5% Goat serum solution (ab7481, Abcam, Cambridge, UK) for 30 min. Clots were then stained with a 1:50 dilution Citrulline Monoclonal Antibody (2D3.1) (MA5-27573, ThermoFisher Scientific) and incubated for 1 h. Following another 3× PBS wash to remove unbound antibodies, samples were then stained with 1:500 dilution Goat Anti-Mouse IgG Secondary antibody conjugated to AlexaFluor 488 (A327273, ThermoFisher Scientific) and incubated for 1 h. Slides were washed 3× with PBS to remove unbound antibody, allowed to dry, and mounted with a glass coverslip. Samples were viewed with a Zeiss Axio Observer 7 Microscope with a Plan-Apochromat 63x/1.4 oil DIC M27 objective (Carl Zeiss Microscopy, Munich, Germany). The excitation wavelength for AlexaFluor488 was set at 450 to 480 nm and the emission wavelength at 499 to 529 nm. Three representative micrographs per sample were analyzed for relative mean fluorescent intensity (MFI) using ImageJ (Version 1.52p). Images were calibrated to scale, and a global threshold (20 pixel cut-off) applied to all analyzed micrographs.