Result

“In hbsmc, the anti CaMKII visualized with a secondary antibody labeled with Alexa 488 showed a punctuated distri-bution near the cell membrane and through the cytoplasm similar to that observed with an anti-CaMKII obtained from a different company. The negative control showed no specific and very low unspecific labeling (nor shown). At this moment, it is difficult to evaluate the antibody specificity due the lack of a western blot against proteins extract-ed from human detrusor tissue. “ - Ricardo Peneda, University of Colorado Anschutz Medical Campus.

Cell Line: Human Bladder Smooth Muscle

cells

Method of validation: Immunofluorescence

Primary Antibody: STJ91990 Anti-CaMKII

Secondary Antibody Chicken anti-rabbit Alexa488

Reference antibody: CaMKII (provided by Abcam)

Dilution ratio: 1:100

Protocol

Treatment of Materials: Freshly dissociated human bladder smooth muscle cells (hbsmc). Seeded on PLL covered glass coverslips. Fixed with 4% PFA on buffer phosphate-saline for 20 min. Rinse 3X with PBS. Blocking: BSA 1%, Triton 0.1%, Tween 20 0.05% in PBS for 20 min. Primary antibody incubation: primary antibody in blocking solution and incubated at 4C̊ for overnight. Wash: PBS 3X 10 min each Secondary antibody incubation: secondary antibody in blocking solution and incubated at RT for 2 hrs. Wash: PBS 3X 10 min each Visualization: Coverslips mounted in ProLong Antifade on glass slides. Dry overnight at RT. Confocal imaged.

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