Opal 4 color fluorescent ihc kit

After standard slide preparation as described above, slides were incubated with chicken anti-MENA^INV (0.25 μg/mL, generated in the lab of Dr. John S. Condeelis) in a blocking buffer for 60 min at room temperature. Samples were washed three times in 0.5% PBST and incubated with an HRP-conjugated IgG anti-chicken secondary antibody for 60 min at room temperature. After washing, slides were incubated with biotinylated tyramide (Perkin Elmer; Opal 4-color Fluorescent IHC kit) diluted at 1:50 in amplification buffer for 10 min. After washing, slides were incubated with spectral DAPI for 5 min and mounted with ProLong Gold antifade reagent (Life Technologies). The slides were imaged on the Pannoramic 250 Flash II digital whole slide scanner, using a 20 × 0.75NA objective lens. Tissue suitable for scanning was automatically detected using intensity thresholding.

For multiplex immunohistochemical staining, anti-PD-L1, PD-L2 antibody described above and Opal 4-Color fluorescent IHC kit (PerkinElmer, Waltham, MA, USA) were used. PD-L1 was optimised using Opal 470 Fluorophore (red), and then PD-L2 was optimised using Opal 520 Fluorophore (green) according to the manual of the Opal IHC kit. Finally, VECTASHIELD mounting medium with DAPI (Vector) was used to stain nuclei.

For immunofluorescent multiple immunostaining, tissue samples were fixed in 10% buffered formalin and embedded in paraffin wax. In order to investigate cells expressing mesothelial markers in the adhesion tissues, triple fluorescent immunostaining with antibodies for PDPN (ab11936, Abcam), WT-1 (ab89901, Abcam) and α-SMA (ab124964, Abcam) was performed. To investigate proliferation of PDPN-positive cells in damaged ceca and adhesion tissues, double immunostaining was performed with antibodies against PDPN and Ki-67 (#12202 Cell signaling). In order to investigate whether neutrophils and/or mesothelial cells express TGF-β1, IL-6, and/or TNF-α, double immunostaining of TGF-β1, IL-6, and TNF-α in combination with Ly6G or α-SMA was performed. Antibody against for TGF-β1 (ab170874) was purchased from Abcam, and antibodies against for IL-6 (21865-1-AP) and TNF-α (17590-1-AP) were from Proteintech Group Inc. Fluorochrome labeling was performed with Opal 4-color fluorescent IHC kit (NEL820001KT, PerkinElmer) and viewed under a Zeiss LSM780 confocal microscope and documented using LSM780 software.

Immunofluorescence (IF) staining was performed using Opal 4-color fluorescent IHC kit (NEL800001KT, Perkin-Elmer, Waltham, MA, USA). Following antigen retrieval formalin fixed and paraffin embedded (FFPE) sections were incubated with protein block (RE7102, Novolink™ Polymer Detection System, Leica Biosystems, Wetzlar, Germany) for 10 min. For multiplex staining antibodies were used sequentially, first, anti-CD20 (1:100) (M0755, DAKO, Glostrup, Denmark), second anti-PD1 (used undiluted) (ab52587, Abcam, Cambridge, UK) and lastly anti-Ki67 (1:100) (M7240, DAKO) antibodies were incubated with tissue sections for 30 min at room temperature. Following incubation with HRP conjugated goat anti-mouse-IgG (1:200) (P0447, DAKO) for 1 h at room temperature slides were washed and incubated for 10 min at room temperature in the dark with Opal 520 (dilution 1:50) for anti-CD20, Opal 670 (1:100) for PD1 and Opal 570 (dilution 1:50) for Ki67. After additional washes nuclei were counterstained with DAPI (D1306, ThermoFisher). A negative control slide, incubated with mouse IgG1 (X0931, DAKO) was included in each staining run. Images of the stained slides were recorded using NanoZoomer-XR Digital slide scanner C12000–01 (Hamamatsu Photonics, Hamamatsu, Japan) and the images were pseudo-coloured and merged by using ImageJ analysis software [16 (link)].