Las af scan software

Three‐dimensional image stacks (1 μm step size, 40× objective) of 4G8/Clec7a/Iba1‐stained sections were taken on a Leica TCS SP5 confocal laser scanning microscope controlled by LAS AF scan software (Leica Microsystems, Wetzlar, Germany). Per animal, 10 serial coronal sections and three regions per section were used for analysis. The number of mice per group analysed was as follows: female APP23 n = 8, female APP23p40^−/−n = 10, male APP23 n = 10 and male APP23p40^−/−n = 8.
The expression levels of Iba1 and Clec7a, 4G8 positive plaques, radial intensity profiles, cell numbers and distances to the nearest plaque (Figs 2C, 5C and 7C) were quantified from maximum projections of the confocal stacks. The quantification was performed in an automated manner using custom‐written ImageJ macros (segmentation) 59 and python scripts (radial profiles and other statistics), which can be found on GitHub (https://github.com/ngimber/AlzheimersWorkflow). Data were pooled by calculating the median from all images per animal and plotting the mean and SEM of all animals from one group. Data that are displayed as a histogram were binned image‐wise. Histograms and radial intensity profiles were normalised (divided by its own integral) and then pooled as mentioned above.

After the last imaging session, the animals were sacrificed using CO2, and perfused with PBS. The brain was harvested and fixed in 4% paraformaldehyde. Free floating sections of 40um were cut with a cryostat, and stained with anti-Iba1 (1:500, Wako, 019-19741), anti-GFP (1:500, Abcam, AB290), anti-RFP (1:200, Rockland, 600-406-379) to visualize myeloid cells, with anti-CD68 (1:100, AbD Serotec, MCA1957) for activation, or for aβ with 4G8 (1:500, Covance, Sig39220-500). Stereology was performed using a Stereo Investigator system (MicroBrightField) and DV-47d camera (MicroBrightField) mounted on a BX53 microscope (Olympus). Stereological quantification of Iba1+, GFP+ and RFP+ cells were performed using the Optical Fractionator method (MicroBrightField). Quantification of 4G8 + plaques was performed using the Area Fraction fractionator method (MicroBrightField). Percentage of activated myeloid cells around plaques was analysed with ImageJ from maximum intensity images compiled from a stack, taken with a Leica TCS SP5 confocal laser scanning microscope controlled by LAS AF scan software (Leica Microsystem, Wetzlar, Germany).

Cells were seeded at a density of 50,000 cells per well on 12 mm coverslips. After treatment, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in PBS for another 20 min and blocked with 3% bovine serum in PBS for 1 h. The primary antibodies (anti-ASC, AdipoGen, AG-25B-0006, 1:500; anti-IBA1, Wako 019-19741, 1:1000) were added overnight at 4 °C. Subsequently, cells were incubated with the fluorescent secondary antibodies (Alexa Fluor 568-conjugated anti-rabbit IgG, 1:500, Invitrogen, A11011; 488-conjugated anti-rabbit IgG, Invitrogen A21206) for 3 h at room temperature. Cell nuclei were counterstained with DAPI (Roche, 10236276001) and coverslips embedded in fluorescent mounting medium (Dako, S3023). Images were acquired using Leica TCS SP5 confocal laser scanning microscope controlled by LAS AF scan software (Leica Microsystems, Wetzlar, Germany). Z-stacks were taken and images presented as the maximum projection of the z-stack. The number and size of ASC specks was assessed using ImageJ software as described before [14 ].