Biotin labeled horse anti mouse antibody

To detect denatured collagen, 7 µm longitudinal cryosections were stained using
col2-3/4m antibody, using a previously described protocol.^{21 (link)}
Briefly, sections were dried for 90 minutes at 37°C, fixed for 5 minutes
in 3.7% 0.1 M phosphate-buffered (pH 7.4) formaldehyde and then rinsed
extensively in a large volume of PBS. Sections were dipped in 0.1% tween PBS
incubated with 1% hyaluronidase (testicular, Type I-s, EC 3.2.1.35,
Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 37°C to enhance the
permeability of the extracellular matrix by removing PGs. Afterwards, they were
incubated in 10% normal horse serum for 30 minutes to block nonspecific staining
and incubated overnight at 4°C with 1/20 col2-3/4m antibody. The next day,
sections were incubated in biotin-labeled horse anti-mouse antibody (1/400, IgG
(H + L), produced in horse, Vector Laboratories, Inc., Burlingame, CA, USA) for
1 hour at room temperature. Finally, they were incubated with streptavidin 555
reagent (streptavidin, Alexa Fluor 555 conjugate, Invitrogen, Waltham, MA, USA)
for 30 minutes. To detect nuclei, they were counterstained with 1:1000 DAPI
(Thermo Fisher, Waltham, MA, USA). After each preparation step, sections were
rinsed with PBS. After mounting with mowiol, stained sections were digitized at
10× (Zeiss Axiovert 200M, Carl Zeiss, Oberkochen, Germany).