Fixation of the scaffolds was performed by incubation
in 10% formalin solution in PBS at 25°C for 24-48 hours.
The fixed scaffolds were then dehydrated by incubation
in graded alcohol (each alcohol for 20 minutes). After
embedding them in paraffin, they were cut into 5 µm-thick
sections for histological evaluation. H
of detergent efficacy to remove the cells and debris from
the testes. Preservation of glycosaminoglycans (GAGs)
and collagen in decellularised scaffolds were assessed by
alcian blue (Sigma, USA) and Masson’s trichrome staining
(Sigma, USA), respectively. Alcian blue was diluted 1:100
in hydrochloric acid (0.1 M). On these samples nuclear
fast red (Sigma, USA) was used for counter-staining
(12 (link), 13 (link)). Also, preservation of ECM proteins, including
fibronectin, collagen IV, and laminin in decellularised
scaffolds was evaluated by immunohistochemistry
(IHC). Initially, the sections were transferred to a 60°C
oven for de-waxing, then further cleared in xylene. Later,
they were rehydrated by alcohol gradient and washing
in water. Then they were incubated in citrate (10 mM
pH=6.0) for 20 min for antigen retrieval. Then the samples
were permeabilized by triton X-100 for 40 minutes and
incubated with anti-fibronectin (mouse monoclonal IgG,
Elabscience Biotechnology Inc., USA), anti-collagen IV
(mouse monoclonal IgG, Elabscience Biotechnology Inc.,
USA), and anti-laminin (rabbit polyclonal IgG, Abcam,
USA). The secondary antibody was Alexa Fluor 488 (goat
anti-mouse IgG, Invitrogen, USA) and Texas Red (Goat
anti-rabbit IgG, Abcam, USA). Photomicrographs were
taken with an Olympus microscope (Olympus, Center
Valley, PA, USA).
in 10% formalin solution in PBS at 25°C for 24-48 hours.
The fixed scaffolds were then dehydrated by incubation
in graded alcohol (each alcohol for 20 minutes). After
embedding them in paraffin, they were cut into 5 µm-thick
sections for histological evaluation. H
of detergent efficacy to remove the cells and debris from
the testes. Preservation of glycosaminoglycans (GAGs)
and collagen in decellularised scaffolds were assessed by
alcian blue (Sigma, USA) and Masson’s trichrome staining
(Sigma, USA), respectively. Alcian blue was diluted 1:100
in hydrochloric acid (0.1 M). On these samples nuclear
fast red (Sigma, USA) was used for counter-staining
(12 (link), 13 (link)). Also, preservation of ECM proteins, including
fibronectin, collagen IV, and laminin in decellularised
scaffolds was evaluated by immunohistochemistry
(IHC). Initially, the sections were transferred to a 60°C
oven for de-waxing, then further cleared in xylene. Later,
they were rehydrated by alcohol gradient and washing
in water. Then they were incubated in citrate (10 mM
pH=6.0) for 20 min for antigen retrieval. Then the samples
were permeabilized by triton X-100 for 40 minutes and
incubated with anti-fibronectin (mouse monoclonal IgG,
Elabscience Biotechnology Inc., USA), anti-collagen IV
(mouse monoclonal IgG, Elabscience Biotechnology Inc.,
USA), and anti-laminin (rabbit polyclonal IgG, Abcam,
USA). The secondary antibody was Alexa Fluor 488 (goat
anti-mouse IgG, Invitrogen, USA) and Texas Red (Goat
anti-rabbit IgG, Abcam, USA). Photomicrographs were
taken with an Olympus microscope (Olympus, Center
Valley, PA, USA).