Slow fade anti fade dapi reagent

To detect neuronal apoptotic cells, sections were treated with 0.1 M citrate buffer at 90 °C (pH 6.0) for antigen retrieval. TUNEL assay was used with an in-situ cell death detection kit (Roche, Mannheim, Germany) followed by incubating with mouse monoclonal primary anti-NeuN antibody (MAB377; Millipore, US) at 4 °C overnight. Sections were then incubated with donkey anti-mouse IgG Alexa Fluor® 594-conjugated secondary antibody (Thermofisher, US) for 30 mins. Sections were mounted by slow fade® anti-fade DAPI reagent (Lifetech, US).
To quantify the degree of apoptotic cell death in the cortex and hippocampus, ten coronal sections from rostro-to-caudal region of the cortex and hippocampus were examined and TUNEL-positive cells were manually counted in a defined sampling region. One sampling region of interest from the hippocampal CA1 and two sampling regions in the cortex were defined. Only TUNEL-label cells colocalized with condensed nuclei staining were counted. The number of TUNEL-positive cells was the sum of apoptotic cell counts from all sample fields in the quantified sections of one embedded block. The number of TUNEL-positive cells reported was the mean of n number in each group.

BV2 cells were seeded on glass chamber slides at a concentration of 1 × 10⁴ cells/well overnight, and then washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at RT. The slides were rinsed with PBS and incubated with 0.05% Triton X-100 in PBS for 15 min at RT, followed with blocking solution (90% PBS, 10% goat serum, and 0.05% Triton X-100) at RT for 1 h. Slides were then incubated with mouse anti-Iba-1 (1:500, Abcam, Cambridge, MA, USA) and rabbit anti-AdipoR1 (1:500, Abcam, Cambridge, MA, USA) or goat anti-AdipoR2 (1:500, Abcam, Cambridge, MA, USA) at 4 °C overnight. After incubation, the cells were washed with 0.05% Triton X-100 in PBS three times and incubated with appropriate Alexa-Fluor-conjugated secondary antibody (Thermo Fisher Scientific, USA) at RT for 1 h. The coverslips were mounted with by slow fade® anti-fade DAPI reagent (Lifetech, US). The fluorescent images were captured with a Nikon Eclipse Ecliose NiU microscope (Nikon Instruments, Melville, NY) and digitized with SPOT software 5.0 (Diagnostic Instruments, Inc. USA).

For immunohistochemistry, mice were anesthetized with Ketamine/Xylazine. Dissected brains were fixed overnight in 4% paraformaldehyde at 4 °C. Fixed brains were dehydrated in gradient ethanol followed by xylene and embedded in paraffin wax. Sections were rehydrated by graded ethanol to water. Antigens retrieval was performed by incubating sections with 10 mM citrate buffer. Endogenous peroxidase was inactivated by hydrogen peroxide solution. Sections were incubated with primary antibodies, goat anti-GFAP (Santa Cruz, US), mouse monoclonal anti-Aβ (4G8, Biolegend), and rabbit anti-Iba-1 (Wako, Japan) at 4 °C overnight. Sections were incubated with either rabbit anti-mouse or rabbit anti-goat (1:200; Dako, Glostrup, Denmark). Brown color staining was developed and counterstained with hematoxylin. Quantification of staining was performed as described previously [58 (link), 61 (link)]. For immunofluorescent staining, sections were incubated with rabbit anti-AdipoR1 (ab70362, Abcam, UK) at 4 °C overnight, followed by Donkey anti-rabbit IgG Alexa Fluor® 594-conjugated secondary antibody (Thermofisher, US) for 30 mins. Sections were mounted by slow fade® anti-fade DAPI reagent (Lifetech, US).