Cryosections were labelled by indirect immunofluorescence using single- and dual-label approaches. A minimum of five separate corneas were examined for each embryonic age studied. For single labels, sections were rehydrated with PBS containing 0.1% Tween20 (Sigma-Aldrich, Poole, UK; PBST) and incubated with primary antibodies overnight at 4 °C. These were the mouse monoclonals B3D6 to chicken fibronectin, M1B4 to chicken tenascin-C and 5C9 to chicken perlecan (all at 5 µg/mL in PBS) [67 (link),68 (link),69 (link)]. Sections were washed in PBST (three changes over 15 min) and then incubated with horse anti-mouse IgG conjugated with Dylight 488 secondary antibody for 6 h (Vector Laboratories, Peterborough, UK; 5 µg/mL). Sections were washed in PBST as above and mounted in Fluoroshield antifade mountant containing DAPI as a nuclear counterstain (Sigma-Aldrich, Poole, UK). Antibodies B3D6, M1B4 and 5C9 were obtained from the Developmental Studies Hybridoma Bank created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242, USA.
Dual labels for fibronectin and tenascin-C were possible due to the different IgG subtypes of the two primary antibodies—B3D6 is IgG2a and M1B4 is IgG1. Sections were incubated with a mixture of these two antibodies (5 µg/mL each) as above, washed and then incubated with a mixture of two isotype specific secondary antibodies: goat anti-mouse IgG2a-Alexa 488 conjugate and goat anti-mouse IgG1-Alexa 594 conjugate (5 µg/mL each; Invitrogen, ThermoFisher Scientific, Paisley, UK) and mounted as described above. Dual labels for fibronectin and type IV collagen used antibody B3D6 as above, mixed with polyclonal rabbit anti-chicken type IV collagen (5 µg/mL; anti-COL4A1 antibody, Antibodies-online, Aachen, Germany), using the same incubation times and procedures as described above. The secondary antibodies were horse anti-mouse IgG-Dylight 488 conjugate and horse anti-rabbit IgG-Dylight 594 conjugate (5 µg/mL each).
Dual labels for fibronectin and tenascin-C were possible due to the different IgG subtypes of the two primary antibodies—B3D6 is IgG2a and M1B4 is IgG1. Sections were incubated with a mixture of these two antibodies (5 µg/mL each) as above, washed and then incubated with a mixture of two isotype specific secondary antibodies: goat anti-mouse IgG2a-Alexa 488 conjugate and goat anti-mouse IgG1-Alexa 594 conjugate (5 µg/mL each; Invitrogen, ThermoFisher Scientific, Paisley, UK) and mounted as described above. Dual labels for fibronectin and type IV collagen used antibody B3D6 as above, mixed with polyclonal rabbit anti-chicken type IV collagen (5 µg/mL; anti-COL4A1 antibody, Antibodies-online, Aachen, Germany), using the same incubation times and procedures as described above. The secondary antibodies were horse anti-mouse IgG-Dylight 488 conjugate and horse anti-rabbit IgG-Dylight 594 conjugate (5 µg/mL each).
Free full text: Click here
