Fluoromont g

IMR-90 fibroblasts at PN16 and PN35 were cultured on precision glass coverslips thickness No. 1.5 H (Marienfeld Superior). Immunofluorescence was performed as indicated in the previous section, using Fluoromont-G (Thermo fisher Scientific) as mounting medium. SIM was performed on a Zeiss LSM 780 Elyra PS1 microscope (Carl Zeiss, Germany) using 63×/1.4 oil Plan Apo objective with a 1.518 refractive index oil (Carl Zeiss) and an EMCCD Andor Ixon 887 1 K camera for the detection. Fifteen images per plane per channel (five phases, three angles) were acquired with a Z-distance of 0.09 μm to perform 3D-SIM images. Acquisition parameters were adapted from one image to one other to optimize the signal to noise ratio. SIM images were processed with ZEN software and then aligned with ZEN using 100-nm TetraSpeck microspheres (ThermoFisher Scientific) embedded in the same conditions as the sample. The SIMcheck plugin in imageJ was used to analyze the acquisition and the processing in order to optimize for resolution, signal to noise ratio, and reconstruction pattern^{65 (link)}.

Formalin-fixed paraffin-embedded LN tissues were cut into 5 μm sections, deparaffinized at 60°C, and serially washed in 100% xylene, 95% ethanol, 80% ethanol, and 70% ethanol before 100% diH₂O baths (Thermo Fisher Scientific). Antigen retrieval was performed for 15 minutes at 110°C in a Borg decloaker (Biocare Medical). Slides were then submerged in 1× PBS for cooling, followed by permeabilization/blocking for 1 hour in PBS (Thermo Fisher Scientific)/bovine albumin (Millipore Sigma)/Triton-X (Thermo Fisher Scientific). Slides were then stained overnight (4°C) with titrated concentrations of primary antibodies. Slides were washed (3 times, 15 minutes each in 1× PBS) and then stained with titrated concentrations of secondary antibodies for 2 hours at room temperature, followed by 3 more washes. Slides were then blocked with mouse and or goat serum for 1 hour, followed by the addition of titrated concentrations of fluorochrome-conjugated antibodies for 1 hour. Slides were then washed 3 times and stained with nuclear stain (JoPro/Thermo Fisher Scientific) for 15 minutes. Coverslips were then mounted using Fluoromont G (Thermo Fisher Scientific). Tissue sections were stained using JoPro, CD3, CD4, PD-1, CD20, Ki-67, and BCL6. Tfh cells were defined as CD3⁺CD4⁺PD1^hi.