High ph target retrieval solution

Multiplexed fluorescent immunohistochemistry was performed using the tyramide signal amplification method with an Opal IHC kit (Akoya Biosciences, Marlborough, MA, USA) according to the manufacturer's instructions. Tissue sections, of thickness 4 μm, were cut from FFPE tumor specimens and then baked at 60 °C onto adhesive glass slides for 30 min before deparaffinization. The primary antibodies used were anti‐human CD8 (clone C8/144b; DAKO), anti‐human T‐bet (Santa Cruz Biotechnology, Dallas, TX, USA), and cytokeratin (clone AE1/AE3; DAKO). A high‐pH target‐retrieval solution (DAKO) was used for antigen retrieval, and immunoactive buffer (Matsunami Glass, Osaka, Japan) was used for antibody stripping. Opal 520, 540, and 650 were used for labeling CD8, T‐bet, and cytokeratin, respectively. A horseradish peroxidase‐labeled secondary detection system (EnVision Plus, DAKO) was used as a catalyst for fluorophore‐conjugated tyramide. Heating at 95 °C for 20 min was performed for primary antigen unmasking and antibody stripping after each fluorescent labeling.

Sections were de-paraffinized and pre-treated by boiling in 10 mM citrate buffer, pH 6.0, or in high pH target retrieval solution (DAKO). After quenching in 3% H₂O₂, slides were blocked in 20% serum species-matched to the secondary antibody. Staining was developed using DAB (Vector Laboratories, Burlingame, CA) or NBT/BCIP (Roche, Basel, Switzerland). Collagen fibers were visualized using 0.1% Sirius Red. Stereological quantification of capillary tumor blood vessels was performed after CD31 and ASMA, PDGFRβ, NG2 or desmin staining, using an eyepiece grid for unbiased counting, as described earlier [32 (link)]. Stereological quantification of CD31-positive vessels was performed using Leica QWin Standard digital image software and values for 10–40 fields of vision (0.09 mm²) were averaged. The fraction of cleaved caspase-3 positive cells or Ki67 positive cells was determined after analyzing 1000 cells from all tumors in each group.

The experimental process of specimen preparation was the same as for a single IHC. Primary antigen retrieval was performed using High pH Target Retrieval Solution (DAKO, Glostrup, Denmark). The primary antibodies were anti-CD1c (mouse monoclonal, clone OTI2F4; Abcam, Cambridge, UK), anti-CD4 (mouse monoclonal, clone 4B12; Invitrogen, Waltham, USA), anti-CD8 (mouse monoclonal, clone C8/144b; DAKO, Glostrup, Denmark), anti-CD19 (mouse monoclonal, clone LE-CD19; Invitrogen, Waltham, USA), anti-CD21 (rabbit monoclonal, clone SP32; Abcam, Cambridge, UK), and anti-CD138 (mouse monoclonal, clone MI15; DAKO, Glostrup, Denmark). For secondary detection, we used a horseradish peroxidase-labelled detection system (EnVision plus; DAKO, Glostrup, Denmark) as a catalyst for fluorophore-conjugated tyramide. Antigen stripping was performed using Immunoactive Retrieval Buffer (pH 6; Matsunami Glass, Osaka, Japan). Tyramide signal amplification was performed using Opal fluorophore reagents (Akoya Biosciences, Marlboro, USA); Opal 520, Opal 540, Opal 570, Opal 620, Opal 650, and Opal 690 were used for CD1c, CD4, CD8, CD19, CD21, and CD138, respectively. DAPI counterstaining was performed using Spectral DAPI solution (Akoya Biosciences, Marlboro, USA).

Double immunofluorescence staining was performed on sections 3 μm thick. After deparaffinization and rehydration, the sections were subjected to heat treatment for 10 min at 95 °C in High pH Target Retrieval Solution (Dako, Glostrup, Denmark) for antigen retrieval. Sections were incubated with anti-activin A antibody (ab56057, 1:200; Abcam, Cambridge, UK), anti-RANKL antibody (ab216484, 1:100; Abcam), anti-vimentin antibody (MAB2105-SP, 280618, 1:100; R&D Systems, Minneapolis, MN, USA), or anti-CD45 antibody (FAB3791R-025, 1:20; R&D Systems) overnight at 4 °C. After washing in PBS, the sections were incubated with Alexa Fluor 488-conjugated goat anti-rat IgG (A11006, 1:500; Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 568-conjugated goat anti-rabbit IgG (A11011, 1:500; Invitrogen), as appropriate, at room temperature for 30 min. After rinsing with wash buffer, the sections were mounted on glass slides, and images were acquired by confocal microscopy (A1; Nikon, Tokyo, Japan).

The rabbit monoclonal anti-human PD-L1 antibodies used were clone 28-8 (Abcam) and clone SP142 (Spring Bioscience). Heat-induced epitope retrieval with High pH Target Retrieval Solution (DAKO) was performed. Endogenous peroxidase activity was blocked by incubating in 0.3% hydrogen peroxidase and 0.1% sodium azide contained 0.01 M phosphate-buffered saline, and the EnVision plus system (DAKO) was used for secondary detection. The final product was visualized by 3-3′ diaminobenzidine.

Tissue sections were deparaffinized in xylene and hydrated to a decrescent series of ethanol until distilled water. Antigen retrieval was performed using high pH target retrieval solution (Dako, Glostrup, Denmark) in a PTLink module (Dako) for 20 min at 96 °C. Slides were then treated with an endogenous-peroxidase blocking solution (S2023; Dako) followed by a protein blocking solution (X0909; Dako) and the primary antibody at 1:1000: anti-Ki-67 Ab, MIB-5, (Dako M7248). The reaction was detected using the HRP labelled polymer (DakoCytomation) and revealed with diaminobenzidine (DAB) chromogen DakoCytomation).