Lsm 510 inverted

All fluorescent images were acquired using a Zeiss LSM510 inverted confocal microscopes. For immunofluorescence analysis, the cells were fixed with 4% paraformaldehyde in PBS and permeabilised using 0.2% Triton X‐100 in PBS before blocking with 3% bovine serum albumin in PBS. Cells were stained with DAPI and Phalloidin‐TRITC (Sigma #P1951) for 2–6 h at RT. The shape and length of CAFs grown within 3D matrices were determined using Volocity software. Images were thresholded based on F‐actin staining and cells identified in an automated manner.

Images of the brain slices with the delivered probes and the antibody staining were acquired using fluorescence microscopy (10x; Zeiss Axio Observer; Oberkochen, Germany) and confocal microscopy (20x; Zeiss LSM-510 inverted; Oberkochen, Germany). Gd(rhodamine-pip-DO3A) and Texas Red 3 kDa dextran were excited at 562/40 nm and emissions were filtered at 624/40 nm. The Alexa Fluor 488 and FITC stained slices were excited at 470/40 nm and emissions were filtered at 525/50 nm.
The detected dose was measured with the normalized optical density (NOD) 35 (link). All pixels with intensities higher than the mean of the control region plus twice its standard deviation were summed for both the control and targeted regions of interest. The sum of the targeted region was subtracted by that of the control region to obtain the NOD. The distribution of the probes was quantified with the coefficient of variation (COV), defined as the standard deviation over the average fluorescence intensity in the targeted region. This was calculated for six slices for each treated brain by selecting regions of interest around the targeted left hippocampus using Matlab® (2016a, The Mathworks, Natick, MA, USA).