Bs966

All tumor tissue staining experiments were performed at the MD Anderson Cancer Center Research Histology Core Laboratory (28 (link)). Tumors were fixed in 10% neutral buffered formalin, paraffin embedded, processed into a 5 μm thick section/slide, and stained with H&E or various antibodies for IHC. For IHC, the slides were deparaffinized, treated with antigen retrieval buffer, blocked with Sniper (catalog BS966, Biocare Medical), stained with primary and the corresponding secondary antibodies, visualized by diaminobenzidine, and counterstained with hematoxylin. The primary antibodies used were as follows: human-specific cytokeratin (CK) 8/18 (catalog M3652, Dako), collagen VI (catalog ab182744, Abcam), thyroglobulin (TG) (catalog BSB 2767, BioSB), CEACAM5/6 (catalog ab22705, Abcam), paired box gene 8 (PAX8) (catalog 379, Biocare), and thyroid transcription factor 1 (TTF-1) (catalog IS05630-2, Dako). Slides were then examined microscopically by a head and neck pathologist using a BX41 Olympus microscope and an Aperio (Leica Biosystems) digital image scanner. The morphology, degree of differentiation (growth pattern, cytologic features, formation of keratin), and extent of inflammation were evaluated from H&E staining results. The IHC staining intensity was graded according to 3 levels: weak, intermediate, and strong.

The plates containing serial sections of the knee joint samples were
immunostained for human cathepsin K and human MMP-9, which are osteoclast
markers and are involved in bone resorption [30 –33 ]. After deparaffinization using xylene
and drysol, the plates were treated with 0.3% hydrogen peroxide/methanol for
blocking. After inactivation of endogenous peroxidase, they were incubated with
Background Sniper (BS966, BioCare Medical, Concord, CA, USA) and then incubated
with rabbit anti-human cathepsin K polyclonal antibody (M189, Takara, Shiga,
Japan; dilution, 1:50) and rabbit anti-human MMP-9 polyclonal antibody
(LS-C95901, Life Span Bioscience, Inc, Seattle, WA, USA; dilution, 1:25, 1:50,
and 1:100), followed by incubation with horseradish peroxidase-conjugated goat
anti-rabbit antibody (Histofine Simplestain, MAXPO(R), 424141, Nichirei
Biosciences Inc., Tokyo, Japan). Next, they were incubated with
3,3’-diaminobenzidine (425011, Nichirei Biosciences Inc.) and counterstained
with hematoxylin. All stained plates and chamber slides were examined using
light microscopy.

Immunohistochemical analysis was performed using anti-MLH1 (PM220, Biocare Medical, Concord, CA), anti-MSH2 (PM219, Biocare Medical), anti-MSH6 (PM265, Biocare Medical), and anti-PMS2 (PM344, Biocare Medical). Samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5 μm sections. Antigen retrieval was performed using 10mM citrate buffer (anti-MSH2) or Tris-EDTA buffer pH 9.0 (anti-MLH1, anti-MSH6, anti-PMS2) heated to boiling for 32 minutes and blocked for endogenous peroxidase with Peroxidazed 1 (PX968, Biocare Medical). Slides were blocked using Background Sniper (BS966, Biocare Medical), then incubated for one hour at room temperature with antibodies diluted 1:100–1:200. Slides were treated with MACH 4 Universal HRP Polymer (M4U534, Biocare Medical), detected with Betazoid DAB Chromogen (BDB2004, Biocare Medical), counterstained with hematoxylin, dehydrated, and cover-slipped. Samples were graded for the absence or presence of nuclear staining of the MMR proteins. Intact staining in the infiltrating immune cells was used as an internal control. A board-certified pathologist who was blinded to the MSI results confirmed all the IHC results.