Lu nv laser launch

The agarose pads with induced MG1665 cells were prepared similar to those in the “Imaging the IbpA protein with wide-field fluorescence microscopy” section. SIM images were acquired on a Nikon N-SIM system equipped with a Nikon SR HP Apo TIRF 100X 1.49NA objective, a Hamamatsu ORCA-Flash4.0 camera (65 nm per pixel), and 488 nm and 561 nm lasers from a Nikon LU-NV laser launch. Cells were identified using DIC to avoid photobleaching. For each 2D-SIM image, nine images were acquired in different phases via the built-in 2D SIM modes. Super-resolution image reconstruction was performed using the Nikon Elements SIM module.

Spleens and portions of intestine were collected at necropsy from mice challenged with HIV-1 and fixed in 4% paraformaldehyde. Tissues (n = 2 per group) were embedded in OCT, sectioned, and stained as previously described^{32 (link)}. The primary antibodies used were rat anti-CD3 (1:500; Abcam, clone CD3-12), rabbit polyclonal anti-CD8α (1:750; Abcam, ab4055), and mouse anti-CD68 (1:1000; Agilent, clone PG-M1). Secondary antibodies used were goat anti-rat-Cy3 (1:1000; Jackson ImmunoResearch), donkey anti-rabbit-Alexa488 (1:750; ThermoFisher), and donkey anti-mouse-Cy5 (1:1000; Jackson ImmunoResearch).
A Nikon A1 spectral inverted confocal microscope with a 40X 1.49 NS oil immersion objective (Nikon) was used to acquire images of the fixed tissue samples. LU-NV laser launch (Nikon) was used to emit lasers at 405 nm (Hoechst), 488 nm (Alexa Fluor 488), 561 nm (Cy3), and 640 nm (Cy5). For each tissue section, 5 images were acquired from different randomly chosen fields of view. NIS-Elements software (Nikon) was used to analyze fluorescence intensity and frequencies of each fluorescent signal.