Rabbit anti parvalbumin

The following mouse monoclonal primary antibodies were purchased from the UC Davis/NIH NeuroMab facility: AnkG (106/36; RRID:AB_10673030), AnkG (106/65; RRID:AB_10675130), PanNav (N419/78; RRID:AB_2493099), β1 spectrin (N385/21; RRID:AB_2315815), Parvalbumin (L114/3; RRID:AB_2651167), AnkR (N388A/10, RRID:AB_2491109). Other antibodies were sourced as follows: mouse anti-PanNav (Sigma-Aldrich K58/35; RRID:AB_477552), chicken anti-MAP2 (Encor cat. CPCA-MAP2; RRID:AB_2138173), rat anti-GFP (Biolegend cat. 338002; RRID:AB_1279414), chicken anti-Pan-Neurofascin (R and D Systems cat. AF3235; RRID:AB_10890736), rabbit anti-βAPP (Thermo Fisher Scientific, RRID:AB_2533902), rabbit anti-Parvalbumin (Novus, RRID:AB_791498), rabbit anti-active Caspase3 (R and D Systems, RRID:AB_2243952). The following antibodies were described previously: rabbit anti-βIV Spectrin SD antibodies (Yoshimura et al., 2016 (link)); rabbit anti-AnkR (Ho et al., 2014 (link)); rabbit anti-Caspr (RRID:AB_2572297; Rasband et al., 1999 (link)). Secondary antibodies were purchased from Thermo Fisher Scientific and Jackson ImmunoResearch Laboratories.

Whole cell lysate protein concentrations were measured by Bradford assay to ensure equal protein loading. Proteins were separated by polyacrylamide gel electrophoresis using 4–20% gradient gels. Proteins were then transferred to 0.2 µm pore size polyvinylidene fluoride membranes. The membranes were blocked in 5% BSA in tris-buffered saline buffer containing Tween 20 (TBST) for 1.5 hours. The membranes were then incubated in primary antibody solutions containing 1% BSA in TBST overnight at 4 °C. Following primary antibody incubation, the membranes were washed 3 times for 10 minute each wash in TBST. After washing, the membranes were incubated in secondary antibody solutions containing 1% BSA in TBST for 1 hour. SuperSignal West Pico chemiluminescence substrate was used for detection. Images were recorded on an Azure C300 system using automatic exposure settings to prevent oversaturation of bands. The following primary and secondary antibodies were used: mouse anti-GAPDH (1:20,000; Novus Biologicals), mouse-anti-Myosin-hc (1:250; Novus Biologicals), rabbit-anti-parvalbumin (1:10,000; Novus Biologicals), horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG (1:1000; Novus Biologicals) and HRP-conjugated goat-anti-rabbit IgG (1:1000; Novus Biologicals). Densitometry was performed in ImageJ using the Gel Analysis tool.