3 3 diaminobenzidine dab chromogen

IHC analysis was conducted to detect GOLM1 and FAM49B expression in HNSCC tissues. TMA sections were firstly deparaffinized and rehydrated. The antigen retrievals of GOLM1 and FAM49B were applied in citrate acid buffer (10 mM, pH 6.0) for 15 min by a microwave. The sections were incubated with a rabbit anti-human GOLM1 polyclonal antibody (1:400 dilution, NBP1-88775, Novus Biologicals, Centennial, CO, USA) and rabbit anti-human FAM49B polyclonal antibody (1:50 dilution, NBP1-88582, Novus Biologicals, USA) at 4 °C overnight. Subsequently, horseradish peroxidase (HRP) conjugated to the goat anti-mouse/rabbit secondary antibody was added to the slides at 37 °C for 30 min of incubation. 3,3′-diaminobenzidine (DAB) chromogen (DAKO, Santa Clara, CA, USA) and nuclear counterstaining with hematoxylin were executed afterward. Human stomach tissue for GOLM1 and human lymph node for FAM49B were regarded as the positive control. Primary antibodies replaced by PBS were regarded as the negative control.

ctDNA was assayed as previously described [40 ]. For immunohistochemistry, heat-induced antigen retrieval was performed in 1× citrate buffer (Thermo Scientific). Samples were blocked in 2% bovine serum albumin in tris-buffered saline/Polysorbate 20 solution and endogenous peroxidase inactivated in 1.5% H2O2. Samples were incubated with primary antibodies, including AGF2 (Abcam), CHD4 (Abcam), and Cullin1 (Cell Signalling Technology). Biotinylated species-specific secondary antibodies were used at 1:300 (Dako) followed by the avidin-biotin-complex (ABC) method prior to visualisation with 3,3′-Diaminobenzidine (DAB) chromogen (Dako). Bright-field microscopy was performed on an Olympus BX-51 microscope. Murine inguinal mammary fat pads were used as normal control tissue.

Immunohistochemical analysis was performed as described previously [33 (link)]. Antigen retrieval was performed by soaking the specimen on slides in 5 M sodium citrate solution in phosphate-buffered saline containing 0.05% (v/v) Tween 20 (PBST) (pH 6.0). The slides were subsequently blocked with serum-free protein block (Dako, 2016–08) for 30 min at room temperature and incubated at 4 °C overnight optimally with rabbit polyclonal antibodies to IFN-γ (ab25101, Abcam Inc., Boston, MA, USA) or IL-6 (21865–1-AP, Proteintech Inc., Rosemont, IL, USA) diluted 1:200 in DAKO antibody diluent. After washing three times with PBST, the slides were incubated with anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase-labeled polymer (DakoCytomation, Glostrup, Denmark) and subsequently visualized by treatment with 3,3′ diaminobenzidine (DAB) Chromogen (DakoCytomation, #K3465) according to the instructions provided by the manufacturer. Nuclei were visualized using Mayer’s hematoxylin (MERCK, 1:1000 dilution in PBST). For mounting, the sections were rinsed with water, dehydrated in graded ethanol (90% ethanol for 30 s × 3 and 100% ethanol for 30 s × 3), cleared in xylene (for 30 s × 2), and sealed using multi-mount 480 (Matsunami, FM48001). Images were acquired and processed digitally.