Vs120 l100

The brightfield images of the chromogenic ALK IHC stainings were digitalized on a NanoZoomer Digital Pathology scanner (Hamamatsu, Japan). For IF we used a Zeiss AxioImager M2 m microscope and an Olympus slide scanner VS120-L100. Each image was taken in both DAPI (Alexa350) and Cy5 (Alexa647) channels. For all images within a figure, the same parameters of acquisition, such as filter set, exposure time, and filter intensities were used.

Images were taken using microscopes (BX51 and SZX16, Olympus) equipped with a CCD-camera (DP71, Olympus), a confocal laser microscope (FV1000, Olympus), a virtual slide microscope (VS120-L100, Olympus) and a microscope (SZX16, Olympus) equipped with an HD-color camera (CSD240, Ikeda) and a recorder (VISK IR-100, Chunichi Denshi).

After immunohistochemistry, whole brain slices were imaged with a 10x objective using a slide scanner (Olympus, VS120-L100). Images acquired in the virtual slide format (.vsi) were opened and analyzed using the QuPath software [2 (link)]. DAPI signal was used to draw regions of interest (ROI) in the ipsilateral and contralateral hippocampal formations using a drawing tablet. The area covered by the fluorescent signal within the ROI was measured using thresholding algorithms in Fiji [61 (link)]. The algorithm was chosen to fit the observed signal as accurately as possible. Depending on signal distribution and intensity, either Triangle, Moments or Otsu algorithms were selected for analysis.
Identification of individual Aβ plaques and microglia based on their size was performed on QuPath after signal thresholding using dedicated algorithms. For analysis of signal coverage at the level of Aβ plaques, we defined a ROI including a 5-μm peripheral rim around the Aβ signal using QuPath. The area positive for the signal of interest (Iba1, CD68 or HT7) was obtained by thresholding and the measured surface was divided by the total area of the ROI to determine the percentage of coverage.
High-resolution images of the brain tissue with fluorescent immunostainings were taken either with a Leica DM5500 microscope or with a confocal Zeiss LSM700 microscope.

For anatomical analysis of optic fiber- or GRIN lens positions, mice were transcardially perfused with a 4% paraformaldehyde (PFA) solution. The brains were post-fixed in PFA overnight and then transferred to 30% sucrose in phosphate-buffered solution for dehydration. Coronal brain sections of 40 µm thickness were prepared using a HM450 sliding microtome (Thermo Fisher Scientific, Waltham, MA, USA). Slices were mounted on Superfrost Plus slides (Thermo Fisher Scientific) and embedded in Fluoroshield mounting medium containing DAPI (Sigma-Aldrich) to stain cell nuclei. Slices were imaged with a slide scanning fluorescent microscope VS120-L100 (Olympus) with a 10 x /0.4 NA objective, or with a confocal microscope (Leica SP8). Brain atlas overlays are taken from Franklin and Paxinos, 2016 and were fit to the brain section image using scaling and rotations in Adobe Illustrator (Adobe, San Jose, CA, USA). Where indicated, registration of brain section images was performed onto the Allen Brain Atlas using an open-source ABBA alignment tool for FIJI (https://github.com/BIOP/ijp-imagetoatlas), developed at the Bioimaging and Optics Platform (BIOP) at EPFL (Chiaruttini et al., 2022 (link)). The majority of brain structure names and their abbreviations follows Franklin and Paxinos, 2016 ; their correspondence to the Allen brain atlas are given in Figure 7—source data 2.

Lungs were flushed with 1 ml neutral 4% buffered formalin solution. The left lobes of lungs were then embedded in paraffin and stored overnight at 4 °C, sectioned, and stained with periodic-acid Schiff (PAS) reagents (Sigma) according to standardized protocols. Sections were imaged on a slide-scanning microscope (VS120-L100, Olympus, Tokyo, Japan) and analyzed with Fiji software (NIH, Bethesda, MD USA). Mucus score was quantified as the average number of mucus-producing goblet cells per unit length of bronchi circumference. The inflammation score for each mouse was quantified as the inflamed surface area minus the area of the blood vessel lumen, normalized to the average inflamed surface area minus the area of blood vessel lumen in naïve mice.