Supplementation of cryoprotectant with Pinus massoniana bark

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Cysteine and chlortetracycline (CTC) were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). CTC staining solution (750 μM CTC in 20 mM tris (hydroxymethyl) aminomethane hydrochloride, pH 7.8) was supplemented with 130 mM NaCl and 5 mM cysteine. The fixative consisted of 12.5% (v/v) glutaraldehyde in 1 M tris (hydroxymethyl) aminomethane hydrochloride buffer, pH 7.8. The CTC staining solution was prepared daily and maintained in the dark at 4°C until use.Progesterone, fluorescein isothiocyanate-labeled peanut (Pisum sativum) agglutinin (FITC-PSA), avertin and carprofen were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, US). Earle's balanced salt solution (EBSS), phosphate-buffered saline (PBS) and penicillin-streptomycin were obtained from Thermo Fisher Scientific, Inc (Waltham, MA, US). Sodium pyruvate and triethanolamine were purchased from Amresco, Inc (Solon, OH, US). Hoechst 33258 and polyvinylpyrrolidone-40 (PVP-40) were obtained from Shanghai Maokang Biotechnology Co., Ltd (Shanghai, China). Bovine serum albumin (BSA)andglutaraldehyde were purchased from Sangon Biotech Co. (Shanghai, China). Acridine orange was obtained from Polysciences Inc (Warrington, PA, USA). All other chemicals were reagent grade.
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