Plan apochromat 20 0.8 objective

Paraffin-embedded liver/tumor
tissue sections were deparaffinized, rehydrated, underwent heat-induced
antigen retrieval, and then incubated with primary Abs. The primary
Abs types used for detection of Ki-67, F4/80, Gr-1, and CD8alpha were
SP6 (Abcam), BM8 (Biolegend), RB6F8C5 (Biolegend), and D4W2Z (Cell
Signaling Technology), respectively. ImmPRESS anti-rat Ig, ImmPRESS
anti-rabbit Ig, polymer detection kits, DAB peroxidase substrate kit,
(Vector laboratories) liquid permanent red substrate (Dako), and Hematoxylin
Gill II (Leica) were used for detection and visualization. The images
were captured using an automatic digital slide scanner Pannoramic
MIDI with a Plan-Apochromat 20×/0.8 objective (3D HISTECH) by
the Pathology Core Laboratory of NHRI.

Tissues were fixed by fresh
10% formaldehyde overnight and then
underwent tissue processing for paraffin embedding, and 5 μm-thick
sections were prepared by the Pathology Core Laboratory of NHRI. The
tissue sections were stained with hematoxylin and eosin (H&E)
to check cellular and tissue features. Hematoxylin precisely stains
nuclear components a purplish blue, while eosin stains cytoplasmic
components pink. The sections were also immunostained with primary
antibodies specific to UCP1 and F4/80 following the standard protocol.
Histological images were captured using an automatic digital slide
scanner Pannoramic MIDI with a Plan-Apochromat 20×/0.8 objective
(3D HISTECH Ltd., Budapest, Hungary) by the Pathology Core Laboratory
of NHRI.