Anti mouse secondary antibodies

Cells and tissues were lysed in a RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) with protease inhibitor and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged. The protein solution obtained by centrifugation were isolated by 10% SDS-PAGE electrophoresis and then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were sealed with 3% bovine serum albumin (BSA) dissolved in Tris-buffered saline buffer with 0.1% Tween-20 (TBST) for two hours, then incubated overnight at 4 ℃ with primary antibodies. After washing 3 times with TBST for 10 minutes each, the membranes were incubated at room temperature for 2 hours with the corresponding secondary antibody. Next, the membranes were washed with TBST for 3 times, 10 minutes each. The relative quantitative detection of protein was performed by using an electrochemiluminescence (ECL) kit (Pierce, Rockford, IL, USA). The antibodies used for the analysis were shown below: mouse anti-Snail (Cell Signaling Technology, Danvers, MA, USA), mouse anti-E-cadherin, anti-N-cadherin and anti-Vimentin (BD Biosciences, San Jose, CA, USA), mouse anti-β-actin and anti-mouse secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

After receiving
the previously described therapy, all of the animals were killed.^{12 (link)} The hippocampus region of the mice’s
brain was carefully removed after they had been beheaded and was then
promptly transferred to RNA later solution and PBS (1:1) on ice. After
homogenizing the hippocampus brain in a solution of total protein
extraction reagent, the tissue supernatants were unruffled and kept
at −20 °C for further investigations. The protein concentration
was determined using the Bio-Rad protein estimation assay, and the
absorbance at 595 nm was measured. Gel electrophoresis employing SDS-PAGE
12–15% was carried out after normalizing all sample proteins
to 30 g/group. The run’s operation conditions were kept at
50 mA for the first 20–30 min. For the following 1.5–2
h, until the run was finished, they were switched to 120 V. Proteins
from the gel were then transferred to a poly(vinylidene fluoride)
(PVDF) membrane using semi-dry transblotting, according to Santa Cruz
Biotechnology in the United States (Bio-Rad). Among the mouse-derived
primary antibodies used were those against SYP, PSD-95, p-Akt, NF-kB, β-actin,
IL-1, and TNF-α from Santa Cruz, California, in the United States.
Anti-mouse secondary antibodies from Santa Cruz, California, in the
USA, were used in conjunction with these primary antibodies. The results
were used to produce X-ray films.^{12 (link)}