Immunosaver

Diagnostic formalin-fixed, paraffin-embedded tissue blocks obtained from patients with sarcoidosis were provided by Kyushu University Hospital. Paraffin sections (5 μm in thickness) were deparaffinized and boiled with ImmunoSaver (#097-06192, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 45 min. After blocking with 5% BSA (#A2153, Sigma-Aldrich Co., St. Louis, MO, USA)/PBS for 30 min at room temperature, sections were incubated with primary antibodies overnight in a humidified chamber at 4 °C. After incubation with secondary antibody for 1 h at room temperature, sections were mounted in VECTASHIELD Mounting Medium with DAPI (#H-1200, Vector Laboratories, Burlingame, CA, USA). The sections were analyzed, using an LSM700 (Carl Zeiss, Oberkochen, Germany). The following antibodies were used: Rat anti-human CD3 (#ab11089) and Alexa Flour 555-conjugated donkey anti-rat IgG (#ab150154) from Abcam, Cambridge, UK; Rabbit anti-human PD-1 (#86163T), Rabbit anti-human TIM-3 (#45208T), Rabbit monoclonal IgG isotype control (#3900S), and Alexa Flour 488-conjugated goat anti-rabbit IgG (#4412S) from Cell Signaling Technology, Danvers, MA, USA.

Tumor specimens were obtained from 38 consecutive cases from 2013 to 2017 of hypopharyngeal cancer at Miyagi Cancer Center (Natori, Japan), in which patients underwent biopsy before treatment. All of the cases were pathologically diagnosed as squamous cell carcinoma. Immunostaining of formalin-fixed paraffin-embedded tissue was performed as previously described^{8 (link)} using the following antibodies: anti-CD271 (1:2500; C40-1457; BD Biosciences), anti-phosphorylated (p)-RELA (1:1500; ab86299; Abcam, Cambridge, UK), and EnVision + Dual Link System-HRP (anti-mouse and anti-rabbit; Dako, Glostrup, Denmark). Heat-induced epitope retrieval was performed by microwaving the sections in target retrieval solution for CD271 staining (pH 9.0; Dako) or Immunosaver (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) for p-RELA staining.

Xenograft tissues were fixed with 4% paraformaldehyde for 4 h and then embedded in paraffin. Sections were then cut to 3–4 μm thickness. For hematoxylin-eosin (HE) staining, deparaffinized sections were stained with Carazzi’s hematoxylin for 10 min, followed by staining with eosin Y for 10 min. For immunofluorescent staining, deparaffinized sections were boiled in ImmunoSaver (Fujifilm Wako) at 98°C for 60 min for antigen retrieval. After blocking with Blocking One Histo (Nacalai Tesque, Inc.) for 60 min, sections were incubated with primary antibodies, including anti-human vimentin (Invitrogen, Carlsbad, CA, USA; cat. no. MA5-11883; 1:50 dilution) and anti-phospho-CREB (Cell Signaling Technology; 1:200 dilution), diluted in Signal Enhancer HIKARI (Immunostain Solution A; Nacalai Tesque, Inc.) overnight at 4°C. After washing, sections were incubated with secondary antibodies (AlexaFluor 568-conjugated goat anti-rabbit IgG [Thermo Fisher Scientific; 1:400 dilution] and AlexaFluor 488-conjugated goat anti-mouse IgG; 1:400 dilution]) and DAPI (5 μg/mL) diluted in Signal Enhancer HIKARI (Immunostain Solution B; Nacalai Tesque, Inc.) for 60 min at room temperature.