Ab12866

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.

Rat primary cortical neurons were stimulated at days in vitro (DIV) 5 with 50 ng/ml BDNF for 1 h. Cells were fixed with 4% PFA and processed for imaging as described before (Muddashetty et al., 2011 (link)). In brief, cells were permeabilized using TBS₅₀T (0.3%) [50 mM Tris-Cl (pH 7.4) + 150 mM NaCl + 0.3% TritonX-100]. This was followed by treatment with Tris-Glycine solution (0.5 M Tris and 0.2 M Glycine) for background reduction before blocking with blocking buffer [TBS₅₀T (0.1%) + 2% BSA + 2% FBS]. Primary antibodies were incubated in TBS₅₀T (0.1%) + 1% BSA overnight at 4°C which was followed by washes. Alexa Fluor 488 coupled anti-mouse and Alexa Fluor 555 coupled anti-rabbit secondary antibodies were incubated for 1 h at room temperature. Finally, coverslips were mounted for imaging using Mowiol^® 4-88 mounting media. Images were acquired on FV3000 confocal microscope (Olympus) using Plan Apo 60×, NA 1.42, oil immersion objective. Z-series of 6–10 stacks with XY sampling density of 0.094 μm/pixel were taken at 0.5 micron step size from all dendrites. Imaging conditions were kept constant across experiments. Antibodies against p-ser3-cofilin1 (ab12866), p-thr508-Limk1 (ab38508) and total Limk1 (ab119084) were purchased from Abcam. Total cofilin1 (WH0001072M4) and alpha-tubulin (T9026) from Sigma.