Pierce bca assay reagent kit

20 rats (n=5/group) were treated with NBI/Control pretreatment and CFA/Control pretreatment according to the protocols described above. As adults, 72 hours after their CFA/Control pretreatments, they were deeply anesthetized with 5% isoflurane and then euthanized via decapitation. Spinal cords were hydraulically extracted and lumbosacral (L6-S2) segments were isolated and processed according to ELISA kit instructions. Protein concentrations were determined using the Pierce BCA Assay Reagent Kit (Thermo Fisher Scientific, Rockford, IL). CRFR2 content was quantified using an LSBio kit [Lifespan Biosciences, Seattle, WA]. Samples and serial dilutions of standards were processed according to kit protocols. A two-way ANOVA was used to identify treatment effects with Tukey’s HSD for post hoc pairwise comparisons.

Immediately following completion of the CFS, AFS or NFS protocols described in the previous section, rats were deeply anesthetized with 5% isoflurane, decapitated and bladder and spinal cord tissues obtained and frozen in liquid nitrogen and stored in a −80°C freezer. Subsequently, bladder and spinal cord homogenates were assayed for CRFR1, CRFR2, CRF peptide, urocortin 1, urocortin 2 and urocortin 3 content using commercially available ELISA kits: source for Pierce BCA Assay Reagent kit – Thermo Fisher Scientific, Rockford, IL; source for CRF peptide, CRFR1 and CRFR2 kits – LSBio kits, Lifespan Biosciences, Seattle, WA; source for urocortin 1 and 3 kits – Yanaihara Institute, Inc., Shizuoko, Japan; source for urocortin 2 kit – Cosmo Bio Co, Ltd, Tokyo, Japan.