A10523

To mark PCs, brain sections were incubated with a rabbit anti-calbindin D28K antibody (1:1000, PA1-931, Invitrogen, Waltham, MA) at 4 °C for 24–48 h. After being washed in PBS, sections were incubated with a goat anti-rabbit Cy5 (1:500, A10523, Invitrogen) at room temperature for 3 h. After another rinse with PBS, sections were mounted on slides and cover-slipped with a mounting medium (H-1500, Vector Labs). To identify subtypes of c-Fos positive neurons following the social recognition test, sections were incubated with primary antibodies of guinea pig anti-c-Fos (1:1000, 226308, Synaptic Systems, Gottingen, Germany), rabbit anti-CaMKII (1:200, PA5-99558, Invitrogen) and mouse anti-GAD67 (1:500, MAB5406, Millipore, Burlington, MA) at 4 °C for 24–48 h. After being washed in PBS, sections were incubated with secondary antibodies of goat anti-guinea pig Alexa 488 (1:1000, A11073, Invitrogen), goat anti-rabbit Alexa 555 (1:1000, A21428, Invitrogen) and goat anti-mouse Alexa 647 (1:1000, A21236, Invitrogen) at room temperature for 3 h. After another rinse with PBS, sections were mounted on slides and cover-slipped with a mounting medium (H-1500, Vector Labs). Images were taken with a Zeiss LSM 710 confocal microscope and Zen 2.0 software (Oberkochen, Germany).

Enucleated eyes were fixed in Davidson`s fixative overnight and dehydrated through graded ethanol steps, and embedded in paraffin according to standard protocols. Five micrometer thick sections were deparaffinized, rehydrated to PBS, and stained with Hematoxylin and Eosin (H&E) or Periodic acid–Schiff (PAS) reagents. To visualize the tissue structure of the iris, a de-pigmentation step was included, prior to staining, by treating the rehydrated slides with 10% hydrogen peroxide in PBS overnight. For immunofluorescence staining, an antigen retrieval step was performed by boiling the slides in citrate buffer for 10 minutes. The following primary antibodies were used: rabbit anti-collagen IV (Invitrogen, PA185320), rabbit anti-LOXL1 (Novus Biologicals, NBP182827), mouse anti-fibrillin (Millipore, MAB2641). Antibody detection was performed with Cy5 labeled goat anti-rabbit IgG (Invitrogen, A10523) and goat anti-mouse IgG (Invitrogen, A10524). Fluorescent images were taken using a Leica confocal microscope under 63x oil objective lens.

VAMP2 integrated density in BA–NAc neurons was analysed as a marker of TeTxLC cleavage efficacy. In the 12 TeTxLC mice and 6 mice from each control group, two coronal sections at bregma −1.8 mm underwent immunofluorescence staining using rabbit anti-VAMP2 as primary antibody (1:1000; Ab3347, Abcam) and Cy5 goat anti-rabbit (1:500; A10523, Invitrogen) as secondary antibody. For protocol details, see “Brain histology, injection site validation and immunofluorescence staining”. For each hemisphere per section, an image comprising the entire BA was acquired using a confocal laser scanning microscope at ×20 magnification (for details, see the section “Microscopy”). Separate laser channels were used for VAMP2 (647 nm), eGFP (488 nm), mCherry (555 nm) and DAPI (405 nm). In control mice and TeTxLc mice, respectively, mCherry and EGFP were used to identify BA-NAc neurons and the area where the fluorescent signal was most intense was used for VAMP2 quantification. Working with the images and Fiji ImageJ, representative images were used to create a region of interest (ROI) that corresponded to the typical area covered by fluorescently labelled BA-NAc neurons. This same ROI was then applied to each BA and its mean integrated density for VAMP2 was calculated as the product of mean grey value × area.