2.6 Western blot
Western blot analysis was performed as required by BJP
guidelines(Alexander, Roberts et al., 2018). After the rats were
decapitated, the brains were immediately removed to a prechilled brain
matrix with a 1.0 mm coronal slice thickness (RWD Life Technology,
Shenzhen, China). PrL and IL tissues were harvested on ice guided by the
Paxinos and Watson rat brain atlas(Paxinos & Watson, 1986) and stored
separately in prechilled microcentrifuge tubes at −80°C until assayed.
The tissue was homogenized in RIPA buffer (Solarbio, Cat# R0010, China)
supplemented with protease inhibitors (Solarbio, Cat# P0100, China) and
phosphatase inhibitors (Solarbio, Cat# P1260, China). Protein (45 μg)
was separated by 10% sodium dodecyl sulfate‒polyacrylamide gel
electrophoresis and transferred to polyvinylidene fluoride (PVDF)
membranes (0.45 μm, Millipore, USA). The membranes were blocked with 5%
skim milk for 1 h at room temperature and incubated with primary
antibodies, including anti-β-actin (1:4000; Rabbit mAb, ABclonal, Cat#
AC038, Wuhan, China), anti-β-Amyloid (Aβ) (1:50; Mouse mAb, Santa Cruz
Biotechnology, Cat# sc-28365, RRID:AB_626669), anti-Tau (1:1000; Mouse
mAb, Cell Signaling Technology, Cat# 4019, RRID:AB_10695394),
anti-Phospho-Tau (Ser199) (1:1000; Mouse mAb, Cell Signaling Technology,
Cat# 29957, RRID:AB_2798984), anti-glial fibrillary acidic protein
(GFAP) (1:1000; Rabbit mAb, Cell Signaling Technology, Cat# 80788,
RRID:AB_2799963), anti-glutamate-aspartate transporter (GLAST) (EAAT-1
in humans, 1:1000; Rabbit mAb, Cell Signaling Technology, Cat# 5684,
RRID:AB_10695722), anti-glutamate transporter-1 (GLT-1) (EAAT-2 in
human, 1:500; Rabbit mAb, Abcam, Cat# ab41621, RRID:AB_941782),
anti-ionized calcium-binding adaptor molecule-1 (Iba1) (1:1000; Rabbit
mAb, Cell Signaling Technology, Cat# 17198, RRID:AB_2820254) and
anti-BDNF (1:500; Rabbit mAb, Abcam, Cat# ab108319, RRID:AB_10862052)
in TBST buffer (Tris-buffered saline + 0.1% Tween-20) overnight at 4°C.
The blots were then washed with TBST three times before incubation with
HRP goat anti-mouse IgG (H+L) antibody (1:2000; ABclonal, Cat# AS003,
Wuhan, China) or HRP goat anti-rabbit IgG (H+L) antibody (1:2000;
ABclonal, Cat# AS014, Wuhan, China) for 2 h at room temperature. After
3 × 5 min TBST washes, an ECL Enhanced Kit (ABclonal, Cat# RM00021,
Wuhan, China) was used for detection enhancement, and blots were
visualized using ImageJ software. The results were normalized to the
protein expression level of β-actin.