2.13 Histology and immunohistochemistry
Tissue samples were collected to perform histological assessments. Tumor
tissues or mouse tissues were
immediately fixed with 10% neutral buffered formaldehyde. After
fixation for at least 24 h, the tissues were progressively dehydrated in
a series of graded ethyl alcohol solutions (70, 80, 95 and 100%, v/v),
and embedded into paraffin blocks. For pathological examination, tissue
sections (4 μm thickness) were cut from paraffin blocks of heart, lung,
spleen, liver, kidney, and tumor. Subsequently, the sections were
stained with hematoxylin and eosin (H&E) to indicate nucleus and
cytoplasm, respectively. For immunohistochemical staining, tissue
sections were prepared as described above. The sections were immersed in
a Tris/EDTA buffer (pH 9.0; Servicebio, Wuhan, China) and were heated
for 15 min in a microwave oven. Following antigen retrieval, the
sections were incubated with 3% hydrogen peroxide for 10 min to block
endogenous peroxidase activity. After the inactivation of peroxidase,
the sections were blocked with 3% BSA for 30 min at room temperature.
The sections were incubated with primary antibodies at 4 °C overnight,
washed three times with TBST, and incubated with the corresponding
HRP-conjugated secondary antibody (Invitrogen Corporation, Carlsbad, CA,
USA; RRID:AB_2533965; 1:2000) for 50 min at room temperature in the
dark. After washing three times with TBST, 3,3’-diaminobenzidine
(DAB) was dropped as a chromogen, forming a brown-colored precipitate at
the antigenic sites. Finally, the sections were counterstained with
hematoxylin. All stained slides were dried, mounted with neutral gum,
and examined under a light microscope (Olympus, Tokyo, Japan). Primary
antibodies including PCNA (RRID:AB_303394), Ki67 (RRID:AB_443209),
p-STAT3 (Tyr705) (RRID:AB_1658549), c-Myc (RRID:AB_731658), MMP-9
(RRID:AB_1310463), Survivin (RRID:AB_304564) and ICAM-1
(RRID:AB_870702) were purchased from Abcam (Cambridge, UK). The
immune-related procedures used comply with the recommendations made by
the British Journal of Pharmacology (Alexander et al., 2018).