Glycergel anti fade medium

Mice were perfusion fixed via the heart with 4% paraformaldehyde in PBS. After fixation, the brain was removed, post-fixed for 2 h, dehydrated in EtOH and xylene, and embedded in paraffin wax, enabling 2 μm sectioning using a rotary microtome (Leica). The sections were de-waxed and stepwise rehydrated, before epitopes were retrieved by boiling the sections in TEG buffer: 10 mM Tris buffer with 0.5 mM EGTA (pH 9), or in 10 mM citrate-buffer (pH 6). The epitopes were quenched with 50 mM NH₄Cl in PBS, and unspecific binding was blocked by washing with 1% BSA in PBS with 0.2% gelatin and 0.05% saponin. Sections were incubated overnight at 4 °C with primary antibody diluted in 0.1% BSA in PBS with 0.3% Triton X-100. Primary antibodies are listed in Table 1. For fluorescence visualization of the primary antibodies, AlexaFluor 488- or 555-coupled donkey anti-goat, -rabbit, or -mouse secondary antibodies (Invitrogen) were used, and cell nuclei were visualized using Topro-3 counterstaining (Invitrogen). Sections were mounted with a coverslip in Glycergel anti-fade medium (DAKO) and analyzed using a Leica DMIRE2 inverted microscope with a TC5 SPZ confocal unit using a × 63/1.32 NA HCX PI Apo oil objective.

Mice were perfusion fixed via the heart with 4% paraformaldehyde in PBS. After fixation, the brain was post‐fixed for 2 h, dehydrated, and embedded in paraffin wax, which enabled 2 μm sectioning using a rotary microtome (Leica, Wetzlar, Germany). The sections were de‐waxed and stepwise rehydrated before epitopes were retrieved by boiling the sections in 10 mm Tris buffer (pH 9) with 0.5 mm EGTA. Aldehydes were quenched with 50 mm NH₄Cl in PBS and unspecific binding was blocked by washing with 1% BSA in PBS with 0.2% gelatin and 0.05% saponin. Sections were incubated overnight at 4°C with the primary antibody diluted in 0.1% BSA in PBS added 0.3% Triton X‐100. Primary antibodies are listed in Table 2. Positive control tissues included kidneys, brain, vasculature, and red blood cells (not shown).
The fluorescence visualization of the primary antibodies was performed using AlexaFlour 488‐ or 555‐coupled donkey anti‐goat, ‐sheep, ‐rabbit, or ‐mouse secondary antibodies (Invitrogen). Cell nuclei were visualized using Topro3 counterstaining (Invitrogen). Sections were mounted with a coverslip in Glycergel antifade medium (Dako) and analysed using a Leica DMIRE2 inverted microscope with a TC5 SPZ confocal unit using a 63×/1.32 NA objective. Semiquantitative analysis of immunofluorescence images were performed as described previously (Christensen et al. 2013).