Ab76316

The brain tissue of SN was separated after decapitation, as described in the section of ELISA Experiments (n = 5 for each group). Western blot analyses were conducted with the primary antibodies against mGluR5 (1 : 1000, Ab76316, Abcam), p-Akt (1 : 1000, 9271, CST), Akt (1 : 1000, 9272, CST), p-GSK-3β (1 : 1000, 9336, CST), GSK-3β (1 : 1000, Ab227208, Abcam), p-CREB (1 : 1000, 9198, CST), CREB (1 : 1000, 9197, CST), and β-Tubulin (1 : 500, Ab6046, Abcam), following the similar procedure reported previously [23 (link)].

After transcardial perfusion fixation with 4% neutral formaldehyde, the bilateral adrenal glands were removed, and immunohistochemical staining was performed on 3-μm-thick tissue sections. Adrenal gland sections were deparaffinized, washed three times with PBS, and immersed in 0.01 M citrate buffer (pH 6.0); antigen retrieval was performed at 95°C for 15 min. A solution of 3% H₂O₂ was used to block endogenous peroxidase followed by washing with PBS three times. PBS containing 0.2% Triton X-100 was used to permeate the tissue for 15 min, and non-specific tissue sites were blocked with 10% goat serum for 1 h at room temperature. Then, primary antibodies, including anti-mGluR1 (1:200, ab82211, Abcam) and anti-mGluR5 antibodies (1:200, ab76316, Abcam), were added to the sections, followed by incubation overnight at 4°C. Negative controls were prepared by replacing the primary antibody with PBS. According to the instructions provided in the GTVision^TM III Detection System/Mo & Rb Kit, the sections were treated with an anti-rabbit/mouse HRP-labeled secondary antibody (GK500705, Gene Tech) for 30 min at 37°C and then reacted with DAB and stained for further microscopic analysis. Finally, the sections were stained with hematoxylin for 2 min and dehydrated. The sections were then observed under a light microscope and photographed.