Opal fluorescent ihc kit

For multiplex immunohistochemistry (mIHC) staining, tissues from 5 OSCC samples, 5 oral leukoplakia (OLK) samples and 5 adjacent normal tissues samples were obtained during surgical resection at the Hospital of Stomatology, Sun Yat‐sen University (Guangzhou, Guangdong, P.R. China), and written informed consent was obtained from each patient. The mIHC staining was performed with the Opal fluorescent IHC Kit (NEL811001KT, PerkinElmer).¹⁹ At first, antigen retrieval was performed under high temperature and high pressure for 10 min with Tris‐EDTA (pH = 9.0). Subsequently, the slices were incubated with primary antibody for 16 h (4°C). The primary antibodies were listed as follows: anti‐human CD4 (EP204, CST), anti‐human CD8 (D8A8Y, CST), anti‐human PD‐1 (D4W2J, CST), anti‐human TIM‐3 (D5D5R, CST), and anti‐human LAG‐3 (D2G40, CST). Then the slices were incubated with secondary‐HRP and Opal TSA dyes for 10 min (RT) and 20 min (RT), respectively. Afterward, the other cycles of staining were implemented. The Opal detection fluorophores were as follows: CD4‐Opal 650, CD8‐Opal 620, PD‐1‐Opal 570, TIM‐3‐Opal 540, and LAG‐3‐Opal 520. DAPI was applied for nuclear staining. The immunofluorescence images were captured and analyzed with Vectra 3.0 imaging system and inForm image analysis software (Vectra 3.0; Perkin Elmer).

At sacrifice, the livers containing tumours of each mouse were sampled, fixed in zinc‐formalin and sliced to 4‐µm sections. Immunohistochemistry was performed using the Envision^TM + system (DAKO), and immunofluorescence staining was performed using the Opal Fluorescent IHC Kit (PerkinElmer) according to the manufacturer's instructions. The MI‐77 MW rapid processor (Azumaya) was used for some reactions in the process. Immunohistochemistry was performed using the following antibodies: anti‐Ki‐67 (GTX16667, GeneTex); anti‐CD31 (ab28364, Abcam); anti‐αSMA (ab5694, Abcam); anti‐FAP (ab53066, Abcam); anti‐CD4 (#25229, Cell Signaling); anti‐CD8α (#98941, Cell Signaling); and anti‐FOXP3 (#12653, Cell Signaling). Immunofluorescence staining was performed utilizing the following antibodies: anti‐F4/80 (ab6640, Abcam); anti‐NOS2 (sc‐651, Santa Cruz); anti‐CD163 (ab182422, Abcam); and anti‐VEGF‐A (sc‐152, Santa Cruz). A confocal microscope (FV1000D, Olympus) was used for fluorescence observation. For all the studies, randomly selected fields (100 × magnification) for every large metastasis (ie those> 1 mm²) were analysed using WinROOF version 3.6 (Mitani).

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).