Hplc grade water h2o

2, 5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9AA) were purchased from Sigma-Aldrich (St. Louis, MO). HPLC grade water (H₂O), chloroform (CHCl₃), methanol (MeOH), and phosphate buffered saline (PBS) were purchased from Fisher Scientific (Fair Lawn, NJ), while 100% ethanol (EtOH) was purchased from Decon Labs (King of Prussia, PA). Two different matrix solutions were prepared for positive and negative ion mode analyses. For positive ion mode, 0.5 M DHB in 100% MeOH was used, whereas 10 mg/mL 9AA in 70:30 (v:v) EtOH:H₂O was prepared for negative ion mode analysis. For the multicolor immunofluorescence analysis, primary antibodies BAF8, SC-71, and BF-F3 were purchased in 1 mL supernatant form from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, Iowa). Secondary antibodies Alexa Fluor 350 Goat Anti-Mouse IgG_2b (γ2b), Alexa Fluor 488 Goat Anti-Mouse IgG1 (γ1), Alexa Fluor 555 Goat Anti-Mouse IgM (μ chain), and Prolong Gold antifade reagent were purchased from Life Technologies (Grand Island, NY).