Lsm880 laser

Mouse brain samples were perfused with Tyrode solution followed by PFA, then post‐fixed in 4% PFA for 48 h at 4 °C, and paraffin‐embedded at the University of Michigan's facility using Leica ASP 300 and Tissue‐Tek.^[26 (link),
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^] Sections (5 µm) were obtained using a Leica rotary microtome. For histopathological examination, sections underwent Hematoxylin and Eosin (H&E) staining. Concurrently, DAPI staining was performed for Immunofluorescence (IF) in GFP‐positive cells. Imaging of H&E‐stained sections utilized an Olympus BX53 Upright Microscope, selecting ten random fields per section to encompass tumor heterogeneity. For immunofluorescence, paraffin‐fixed glass coverslips post oncostreams formation was imaged using a Zeiss LSM 880 laser scanning confocal microscope. The resulting data were analyzed using the Zeiss Zen (Blue edition) software, version 2.5.

FRAP experiments for the MAP65-3-GFP were conducted on a LSM880 laser scanning confocal microscope equipped with a 40x water immersion objective (NA 1.2, Zeiss). A certain region of interest was bleached using a 488-nm laser line for 100 iterations with 100% laser power. Fluorescent signal recovery was imaged every 10 s and the average fluorescence intensity of the bleached region was analyzed in the ZEN software.

Neutrophils were seeded on untreated coverglasses using 4-Chamber 35 mm dish with 20 mm microwells (Cellvis LLC Cat # D35C4-20-1.5-N) and incubated at 37 °C for 30 min, then fixed with 3.7% paraformaldehyde for 8 min, permeabilized with 0.01% saponin, and blocked with 1% BSA in PBS. The samples were labeled with the indicated primary antibodies overnight at 4 °C in the presence of 0.01% saponin and 1% BSA. Samples were washed and subsequently incubated with appropriate secondary antibodies for 2 hours at room temperature. The cells were then stained with DAPI and mounted with Fluormount G. Where indicated, F-actin was labelled with 5 µl of a 6.6 µM concentration of Alexa 488- or Alexa 647-Phalloidin in 200 µl PBS with 0.05% Triton X-100. Samples were analyzed with a Zeiss LSM 880 laser scanning confocal microscope attached to a Zeiss Observer Z1 microscope at 21 °C, using a 63× oil Plan Apo, 1.4 numerical aperture (NA) objective. Where indicated, the images were collected using enhanced resolution microscopy (Airyscan) which generates images with substantially increased SNR (signal-to-noise ratio)⁶¹ . Images were collected and fluorescence intensity and colocalization quantified using ZEN-LSM software. Actin colocalization at the edges of granules was analyzed using Manders’ correlation coefficient as described below. The images were processed using ImageJ.

Confocal images of cultured cells and AGs were acquired either by using a Zeiss LSM 510 Meta [Axioplan 2], an Olympus FV1000 microscope set‐up with 100× NA1.4 oil objective or an LSM 880 laser scanning confocal microscope equipped with a 63×, NA 1.4 Plan APO oil DIC objective (Carl Zeiss). RI 1.514 objective immersion oil (Carl Zeiss) was employed.