Bp665 1

Transfected HMC3 cells were fixed with 10% neutral buffered formalin (Fisher Scientific SF100–4) for 30 minutes then blocked and permeabilized for 30 minutes with 10% goat serum (Sigma S26-LITER), 0.1% Triton X-100 (Fisher Scientific BP151–500) in PBS (Fisher BioReagents BP665–1). Primary and secondary antibodies were diluted in the same blocking and permeabilization buffer and incubated at room temperature for 90 minutes. Cells were washed three times in blocking and permeabilization buffer between primary and secondary antibodies, and three times in PBS prior to coverslip mounting with Prolong Glass with NucBlue mounting media (Invitrogen P36981) and high-tolerance No. 1.5 coverglass (ThorLabs CG15KH1). Images were acquired using a Nikon A1R HD inverted confocal microscope with a 60X oil objective and NIS Elements AR software.

All protease-free enzymes were from Seikagaku Corporation Tokyo, Japan unless otherwise specified. Keratanase (KS, from Pseudomonas sp.) digested lower molecular weight KS-GAGs, and Chondroitinase-ABC (C-ABC, from Proteus vulgaris) has been shown to enzymatically cleave N-acetylhexosaminidase linkages of higher molecular weight GAG side chains of chondroitin sulfate (chondroitin-4-sulfate ‘C4S,’ chondroitin-6-sulfate ‘C6S’), dermatan sulfate (‘DS’, often referred to as chondroitin sulfate B), and hyaluronic acid [3]. For the purpose of this study the specimens treated with chondroitinase-ABC will be collectively called ‘CS’ even though hyaluronan could have been affected. C-ABC does not act on keratan sulfate, heparin, or heparan sulfate (Scott and Haigh, 1988 (link)).
The lyophilized enzymes were diluted with 0.1% bovine serum albumin (BSA) (BP675-1, Fisher Scientific, Fair Lawn, NJ) to obtain a concentration of 0.1 IU/ml. All GAG-digestions were performed at a pH of 7.2 and a temperature of 37 °C. The diluted enzymes were stored in a −80 °C freezer to maintain enzyme efficacy and prevent denaturing. Upon each GAG digestion epoch, the specimen was gently rinsed with approximately 80 ml of 1X PBS (BP665-1, Fisher Scientific, Fair Lawn, NJ)