IMMUNOFLUORESCENCE CONFOCAL MICROSCOPY

Coelomocytes were collected from sea urchins 48 hrs after challenge with E. coli. The density of coelomocytes in the final suspension was determined using a Neubauer Cell haemocytometer and adjusted to 1 x 106 cells ml-1 with CMFSW-EI (on ice). The cell suspension (100 l) was pipetted onto glass slides and cells were allowed to settle and adhere to the slide surface for 5 min. Cells were fixed at 17C in 0.5%–1% paraformaldehyde (PFA) in CMFSW-EI in a two-step procedure: (i) in 0.5% PFA for 15 min, (ii) 1% PFA for 15 min. After three washes in CMFSW-I, cells were permeabilised with 0.025% Triton X100 in CMFSW-I for 3 min followed by three washes for 5 min each. Non-specific epitopes were blocked with heat inactivated horse serum for 30 min at 17C before the primary rabbit antisera mix was added for 1 h at 17C. The primary antisera mix contained three polyclonal antibodies, anti-Sp185-66, anti-Sp185-68 and anti-Sp185-71 (each diluted 1:10,000 in CMFSW-I), which targeted the N-terminal-, central- and C-terminal- 185/333 regions. The anti-Sp185/333 antibodies cross react with the He185/333 proteins as described. After 3 washes, slides were incubated for 1 h in the dark at 17C in a cocktail consisting of secondary antibody (mouse anti rabbit IgG-Alexafluor 546 conjugate), actin counterstain (phalloidin-Alexafluor 488 conjugate) and/or nuclear counterstain (Toto; all dyes from Invitrogen). Slides were washed three times in CMFSW-I. Finally, Biomeda Gel Mount (ProSciTech) was added, a coverslip overlaid and sealed with nail polish, and the slides stored at 4C until analysed on the Fluoview 300 laser scanning confocal microscope (Olympus).

Sham Nair 2014