Immunofluorescence procedures, confocal image acquisition
and analysis of CA1 neurons
Slices were incubated as described previously (Petrache et al. 2019),
using GABAAR α5 primary antibody (abcam, raised in
mouse, 1:100) incubated concomitantly with the primary antibody
targeting one of the following: calretinin (Swant, raised in goat,
1:1000), somatostatin (Santa Cruz Biotechnology, raised in rabbit,
1:500), cholecystokinin (Frontier Institute, raised in rabbit, 1:1000)
or CaMKII-α (Invitrogen, raised in goat, 1:100). The secondary
antibodies used were as follows: FITC (Sigma-Aldrich, anti-mouse,
1:200), Texas Red (Invitrogen, anti-rabbit/anti-mouse, 1:500) or Alexa
Fluor 488 (Abcam, anti-goat, 1:500). The sections were counterstained
with the nuclear stain, DAPI (Sigma-Aldrich, 1:1000).
Images were acquired at 63× magnification using a ZEISS LSM 880 confocal
microscope and processed using Zen Black 2009. Collapsed Z-stacks were
imported into Fiji (Image J) as .tif files and split into
individual channels. If needed, the background was removed using theBackground subtraction function in Image J. In the channel
corresponding to the cell staining, the outline of the cells of interest
was drawn manually to obtain regions of interest (ROIs). TheColoc2 plugin was then used to obtain Pearson’s R coefficient as
a measure of colocalisation between the channels corresponding to the
ROIs and to the α5 subunit, and Fisher’s transformation was applied to
convert the coefficients to a normal distribution. The results so
obtained were then averaged separately for wild-type andAppNL-F/NL-F animals, respectively, for each of
the cells of interest. There were no age differences observed during the
analysis, so the data were grouped without any age-dependent
segregation, with ages from 2.5 months to 15 months.