The dityrosine-conjugated Keyhole Limpet Hemocyanin (KLH; 500 μL, 1 mg mL−1) was emulsified with an equal volume of complete Freund’s adjuvant to a final concentration of 0.5 mg mL−1,

and the solution was then intramuscularly injected into New Zealand White rabbits. The rabbits were boosted after 4, 8, and 12 weeks with the same amount of antigen in Freund’s incomplete adjuvant until learn more an adequate antibody generation was achieved. Immunohistochemical studies were performed as previously described [25]. Briefly, grouper at post-hatch day 40–45 were fixed in 10% formalin and embedded in paraffin following a routine procedure. Each 5μm-thick section was mounted on a polylysine-coated slide, deparaffinized in xylene, and rehydrated in descending grades (100–70%) of ethanol. Endogenous Tanespimycin cost peroxidase activity was blocked by 10 min incubation at room temperature with absolute methanol containing 3% H2O2. The sections were sequentially blocked with a power block solution (BioGenex, San Ramon, CA, USA), washed with phosphate-buffered saline (PBS), and incubated with polyclonal rabbit anti-grouper crystallin, dityrosine, or polyclonal rabbit anti-coat protein antibody (1:500 dilution) at 4 °C overnight. The sections were washed twice with PBS, incubated with secondary

antibody (Super Sensitive Polymer-HRP IHC; BioGenex, San Ramon, CA, USA) for 30 min at room temperature. Peroxidase activity was ascertained with 3,3′-diaminobenzidine (used as chromogen) for 10 min. The sections were counterstained with Harris hematoxylin for nuclei, dehydrated, and mounted. Negative controls were performed with Epothilone B (EPO906, Patupilone) preimmune rabbit serum and incubation with PBS instead of anti-grouper crystallin antibodies.

The sections were observed using an Axiovert 40 microscope (Carl Zeiss, Jena, Germany). The images were obtained with an SPOT RT3 camera (Diagnostic, Sterling Heights, MI, USA). cDNA from amino acids 1–231 for the mature crystallin of an orange-spotted grouper was cloned into a pGEM-T vector (Promega, Madison, WI, USA) and subcloned into the pET29a vector (Novagen, San Diego, CA, USA) between the EcoRI and XhoI sites to obtain pET29a-crystallin. The resulting expression vector encoded crystallin with a (His)6 and several extra amino acids at the N-terminus. This vector was transformed into the bacterial host, Escherichia coli BL21 (DE3), for expression driven by T7 polymerase. Induction by 0.5 mM isopropyl-β-thiogalactopyranoside was carried out at 37 °C for 3 h. After undergoing freezing and thawing once, cells were sonicated on ice, and the cleared lysate was obtained by centrifugation at 12,000 rpm for 15 min.