Tissuefaxs imaging software

Muscle tissue was embedded in Tissue‐Tek OCT Compound (Sakura), and 7 μm thick serial sections for staining were cut using a cryomicrotome. To analyse the NADH dehydrogenase activity, we incubated the sections in 0.9 mM NADH and 1.5 mM nitro blue tetrazolium (Sigma) in 3.5 mM phosphate buffer (pH 7.4) for 30 min. To analyse the succinate dehydrogenase (SDH) activity, we incubated the sections for 1 h in 50 μM sodium succinate and 0.3 mM nitro blue tetrazolium in 114 mM phosphate buffer containing K‐EGTA (Sigma). Myosin heavy chain (Myh) immunostaining of muscle tissue sections was performed in the order of fixation, permeation, and incubation with primary antibodies against MyhI, MyhIIa, MyhIIb (Developmental Studies Hybridoma Bank), and laminin (Abcam). For Myh immunostaining, MyhIIa (BF‐32, 1:200), MyhIIb (BF‐F3, 1:200) (Developmental Studies Hybridoma Bank), and laminin (ab11575, 1:200; Abcam) antibodies were used. The images were captured and processed with a Nikon ECLIPSE TE‐2000 U inverted microscope using nis‐elements f software (Nikon), and the myofibre area was measured using imagej software. For muscle histology, the cryosections were stained with Mayer's hematoxylin and eosin (BBC Biomedical). The images were captured using a Nikon ECLIPS TE‐2000 U inverted microscope or tissue faxs imaging software (TissueGnostics).

An Opal Fluorescent IHC Kit (PerkinElmer cat# NEL811001KT) was used to perform mIHC staining. OSCC tissue slices were incubated overnight with primary antibodies at 4 °C. The primary antibodies used were as follows: anti-CD4 (CST cat#25,229), anti-CD19 (CST cat#3574), anti-CD8 (CST cat# 98,941), anti-TCL1A (Santa Cruz Biotechnology cat# sc-393,436), and anti-PNAD (Abcam cat# ab111710). The OSCC slices were incubated with secondary antibody for 10 min at room temperature. After that, all slices were incubated with dye (Opal TSA) for 20 min at room temperature. The Opal detection fluorophores used were as follows: CD8-Opal 690, CD4-Opal 650, PNAD-Opal 620, CD19-Opal 570, and TCL1A-Opal 520. All mIHC images were captured using TissueFAXS Imaging software (TissueGnostics v7.134).

Quadruple immunofluorescence staining of the acetone-fixed cryosections was performed using directly labeled monoclonal and secondary antibodies for increased signal strength (Table S3, Supplementary Materials). Briefly, after rehydration and blocking, the slides were incubated in several steps with primary antibodies overnight at 4 °C and appropriate secondary antibodies for 30 min at room temperature, followed by counterstaining with DAPI. Appropriate isotype controls were stained at the same time. For evaluating the immunofluorescence results, the images were acquired at room temperature using a Z1 Axio Observer microscope equipped with an LD Plan-Neofluar ×20/0.4 objective (Zeiss, Oberkochen, Germany) using TissueFAXS imaging software and quantified with TissueQUEST image analysis software (TissueGnostics, Vienna, Austria).