Ab42824

Rats were given halothane to be anesthetized and then perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.2. Brains were postfixed for 2 h, cryoprotected in 20% sucrose/PB, and then sectioned (25μm) on a freezing microtome. The position of the slices was based on Bregma 2.76mm. Free-floating sections were processed for double-immuno-labeling that rabbit anti-Cofilin (1:100, ab-42824, Abcam) or anti-pCofilin [pS3] IgG (1:100, ab-12866, Abcam), and mouse anti-PSD-95 (1:200, MA1-045, Thermo Fisher Scientific) were used as the first antisera, and Alexa Fluor 555 anti-rabbit IgG and Alexa Fluor 488 anti-mouse IgG (both 1:200; Invitrogen, Carlsbad, CA) in PB as the secondary antisera. Control tissue was processed by the same procedures but without the first antisera. Immunocytochemistry for pCofilin and PSD-95 was examined to three batches of tissue, and each batch was simultaneously processed, and each group contained 2-4 samples. All the groups N, S, C, non-TTI, and TTI contributed their samples to test. Every batch contained tissue from the group C, and quantification of immunoreactive puncta within a batch was normalized to the mean value of the group C for that batch.

Western blot was performed as previously described (15 (link)). Briefly, the protein concentrations of the RSV infection serum samples (15 patients with acute- and convalescence-phase RSV infection, 15 control) and mouse lung homogenates (10 mice with acute- and convalescence-phase RSV infection, 10 control) were determined using a BCA kit (Beyotime Biotechnology, China). Protein samples (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at constant voltage in Tris-glycine buffer, and then transferred onto polyvinylidene difluoride membranes at constant current. The membranes were washed thrice in Tris-buffered saline containing 0.1% TWEEN 20 (TBST), blocked with 10% bovine serum albumin for 1 h at room temperature, and incubated with primary antibodies against triosephosphate isomerase 1 (TPI1; ab28760, Abcam, USA), peroxiredoxin 2 (PRDX2; ab109367, Abcam), bisphosphoglycerate mutase (BPGM; ab97497, Abcam), and cofilin 1 (CFL1; ab42824, Abcam) at 4°C overnight. The next day, membranes were washed three times in TBST and incubated with secondary antibody for 1 h at room temperature. After three washes in TBST, protein signals were visualized with ECL (Beyotime Biotechnology, China) and exposed to X-ray film. The band intensities were quantitated using ImageJ software (Version 2.0; NIH, USA) and normalized to GAPDH.

Cofilin 1 protein expression in bladder cancer tissues and corresponding paracancerous tissues was evaluated by IHC with the 3′, 3′-diaminobenzidine (DAB) kit (ZLI-9017, ZSGB-Bio, Beijing, China), which was performed according to the manufacturer's protocol. Cofilin 1 monoclonal antibody (ab42824, 1:200, Abcam, USA) and goat anti-rabbit HRP antibody (ab136817, 1:400, Abcam, USA) were used.