All immunohistochemistry
procedures were carried out by MicroMorph (Lund, Sweden). The tissue
samples were automatically dehydrated and embedded in paraffin in
a TISSUE-TEK VIP (Miles Scientific) before being embedded in paraffin.
Paraffin sections (4 μm) were prepared and dried in an oven
at 37 °C overnight. The slides were subjected to deparaffinization
and pretreatment antigen retrieval pH 9.0 (Dako, S2367) by boiling
at 100 °C for 20 min and then cooling at RT for 20 min. The slides
were then immersed in PBS for 5 min, following by blocking the sections
with 5% normal goat serum. The rabbit anti-mouse PEG B-47 (Abcam,
Ab51257) primary antibody was diluted 1:1000 in PBS + 5% normal goat
serum for incubation of the sections with the antibody at RT for 1
h. Next, slides were washed 3× with PBS and incubated with the
goat anti-rabbit HRP (immunologic, DPVR110HRP) secondary antibody
for 30 min at RT. Then, slides were washed with Tris buffer 3×,
and sections were incubated with 3,3′-diaminobenzidine for
5 min at RT, followed by hematoxylin counterstain, dehydration, and
mounting of a cover slide.
procedures were carried out by MicroMorph (Lund, Sweden). The tissue
samples were automatically dehydrated and embedded in paraffin in
a TISSUE-TEK VIP (Miles Scientific) before being embedded in paraffin.
Paraffin sections (4 μm) were prepared and dried in an oven
at 37 °C overnight. The slides were subjected to deparaffinization
and pretreatment antigen retrieval pH 9.0 (Dako, S2367) by boiling
at 100 °C for 20 min and then cooling at RT for 20 min. The slides
were then immersed in PBS for 5 min, following by blocking the sections
with 5% normal goat serum. The rabbit anti-mouse PEG B-47 (Abcam,
Ab51257) primary antibody was diluted 1:1000 in PBS + 5% normal goat
serum for incubation of the sections with the antibody at RT for 1
h. Next, slides were washed 3× with PBS and incubated with the
goat anti-rabbit HRP (immunologic, DPVR110HRP) secondary antibody
for 30 min at RT. Then, slides were washed with Tris buffer 3×,
and sections were incubated with 3,3′-diaminobenzidine for
5 min at RT, followed by hematoxylin counterstain, dehydration, and
mounting of a cover slide.