Immunostaining for Extracellular Matrix Components

Samples were permeabilized with 0.1% Triton X-100 in PBS 1×
for 1 h at RT, and then to block nonspecific staining between the
primary antibodies and the tissue, sections were incubated with 1%
horse serum in PBS for 30 min at RT. Primary antibodies were added
against type I collagen (1:100, Merck Millipore), fibronectin (20
μg/mL), produced as previously described,^{27 (link)} and elastin (1:500; Sigma-Aldrich) diluted in 1% horse
serum in PBS + 0.02% (v/v) Tween 20. Samples were incubated overnight
at 4 °C with primary antibodies; then, hydrogels were incubated
with polyclonal goat antirabbit Ig/HRP secondary antibody (1:150 Dako)
(for type I collagen and fibronectin immunostaining) and with polyclonal
rabbit antimouse Ig/HRP secondary antibody (1:150 Dako) (for elastin
staining), both diluted in incubation buffer for 1 h at RT and protected
from light. At the end of the incubation, 0.03% of DAB dissolved in
Tris buffer + 0.02% H₂O₂ to reveal the precipitate.
Lastly, tissues were dehydrated and cleared in xylene and mounted.