Rat anti-mouse CD4 (550280) was from BD Pharmingen (Franklin Lakes, NJ, USA), antibodies against cytokeratin-10 (K10) (sc-31770) and K12 (sc-17101) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); AlexaFluor 488-conjugated donkey anti-goat was from Invitrogen (Carlsbad, CA, USA); Horseradish peroxidase (HRP)-conjugated donkey anti-Rat IgG was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Triton X-100, Tween 20, and pilocarpine were from Sigma-Aldrich Corp. (St. Louis, MO, USA). Diaminobenzidine (DAB) kit and antibody diluent were from DAKO North America (Carpinteria, CA, USA). Antigen retrieval citrate buffer (pH 6.0) was from Invitrogen. Mounting medium (FluorSave) was from Millipore Corp. (Billerica, MA, USA). Lissamine green stain was from Leiter's Pharmacy and Compounding Center (San Jose, CA, USA). Diagnostic tear test (Zone-Quick Thread Tears) were from Showa Yakuhin Kako Co., Ltd. (Tokyo, Japan), distributed by Menicon American, Inc. (San Mateo, CA, USA).
Nitrocellulose membrane and ECL reagents were from Amersham, GE Healthcare (Little Chalfont, UK). Horseradish peroxidase–conjugated donkey anti-rabbit antibody was from Jackson ImmunoResearch Laboratories, Inc. Lacritin was generated in
E. coli from the pLAC plasmid as an intein fusion protein. Expressed lacritin was purified on chitin columns with mercaptoethanol elution that cleaves off the intein tag. Further purification was performed on diethylaminoethanol columns, all as previously described.
4 The recombinant lacritin is approximately 18 kD in size. Purified lacritin is stable, without cleavage or degradation, for 6 months when lyophilized and stored frozen. When reconstituted, it is stable at either 4°C or room temperature for a 24-hour period. A new vial of lyophilized lacritin was reconstituted on each day of the treatment period. Lacritin truncation mutant lacking 25 amino acids from its C-terminus (C-25) was synthesized by previously described methods.
14 Lacritin antibody referred to as
anti-N-65 Lac C-term was produced in rabbits against the C-terminal end of human recombinant lacritin using the N-65 truncation mutant.
11