Uv laser

For rat pituitary we used coronal sectioning of formalin-fixed paraffin-embedded male pituitaries in 4-micron sections. For dewaxing sections were incubated overnight at 52°C. Antigen retrieval was done using the PT-Link system in high pH buffer (DAKO-Agilent). Sections were incubated with primary antibodies overnight at 4°C and then 1 h with secondary antibodies at room temperature. For nuclei staining we used DAPI. Concentrations and conditions of antibody incubations are listed in Supplementary Table 2. Slides were photographed using a confocal microscope TC-SP5-AOBS with a white laser (470–670 nm) and a UV laser (Leica) with LAS AF (Leica Application Suite Advanced Flourescence) software, using serial sections (Z) every 1 μm for the 20X (PL APO 20X/N.A.0.70 CS) objective and 0.3 μm for the 63X objective (oil PL APO 63x/N.A.1.4-0.6 CS). Data were collected using Sequential Mode with the following order: first, Channel 00 (DAPI); second, Channel 02 (Alexa 488); third, Channel 03 (Cy3). Data were collected at resolution 1,024 × 1,024 pixels, with zoom 1x-3x, giving an XY field of a range from 775 × 775 μm till 288.5 × 288.5 μm for objective 20x and of a range of 246 × 246 μm till 90.4 × 90.4 μm for objective 63x. Thus, the final resolution was between 0.75 and 0.28 μm/pixel (20x) and 0.24–0.08 μm/pixel (63x).

After fixation in formalin, FFPE brain tissue blocks containing either the superior frontal gyrus of the frontal cortex or hippocampus (level of LGN) were sectioned at 8 µm and collected onto LCM compatible PET slides (Leica). Sections were stained with cresyl violet to localize regions of interest for LCM [15 (link)] and air dried overnight in a loosely closed container. LCM was used to individually microdissect 10 mm² from the frontal cortex (gray matter layers I–IV) and hippocampal CA1-3, and 4 mm² from the hippocampal DG into LC–MS grade water (Thermo Scientific). Microdissected samples were centrifuged for 2 min at 14,000 g and stored at – 80 °C. LCM was performed at 5X magnification with a LMD6500 microscope equipped with a UV laser (Leica).