Envision dual link system hrp dab

Archival tumor tissue was analyzed by immunohistochemistry (IHC) for PTEN, Beclin 1, and PDGFRb expression. Tumor tissues were deparaffinized and rehydrated through graded alcohols to water. Heat-induced antigen retrieval was done with sodium citrate buffer (10 μM, pH 6.0) for PTEN staining and with Tris/EDTA buffer (pH 9.0) for Beclin 1 and PDGFRβ staining. The slides for staining with anti-PTEN or anti-PDGFRβ antibodies were washed with TBS containing 0.025% Tween 20, and the slides for staining with anti-Beclin 1 antibody were washed with PBS containing 0.1% Tween 20. Before staining, the slides were incubated for 10 minutes with Dual Endogenous Enzyme Block, an endogenous peroxidase inhibitor (EnVision+ Dual Link System-HRP (DAB+); DakoCytomation, Denmark). The slides were then blocked with 10% normal goat serum for 2 hours at room temperature with the diluent for each antibody as specified by the manufacturer. After washing, the slides were stained overnight at 4°C with anti-PTEN (1:100, #9559, Cell Signaling Technology), anti-Beclin 1 (1:1,000, ab62472, Abcam), or anti-PDGFRb (1:100, ab5511, Abcam) antibody. Antibody staining was visualized using EnVision+ Dual Link System-HRP (DAB+) according to the manufacturer's protocol. The slides were read by a clinical pathologist who was blinded to the identity of the patient specimens.

We characterized patient-derived CAFs by testing the expression of SMA (Actin, Smooth Muscle, 1A4; Cell Marque catalog # 202M-94), S100A4 (Recombinant Anti-S100A4 antibody; Abcam catalog # ab124805), TE-7 (Fibroblasts Antibody, TE-7; NOVUS Biologicals, LLC, Centennial, CO, USA; catalog # NBP2-50082), and negative expression of epithelial tumor markers including EpCAM (Ep-CAM/Epithelial Specific Antigen, Ber-EP4; Cell Marque, Rocklin, CA, USA; catalog # 248M-94) as well as CK 8, 18 (Cytokeratin 8 and 18; B22.1 and B23.1 from Cell Marque; catalog # 818M-94). Human uterine fibroblasts (HUF) and NCI-H441 tumor cell lines were used for validation. The percentage of PD-L1 (PD-L1 [Clone 22C3]; Agilent-Dako, Santa Clara, CA, USA; catalog # M365329-1) expression was tested in well-characterized CAFs from both tumors and tumor-adjacent normal samples. The IHC detection kit was procured from Dako (Envision+ Dual-link system-HRP (DAB+), code K4065).

Immunohistochemistry was performed on 498 of the original 563 primary BCs using a Dako EnVision + Dual Link System-HRP (DAB+) (Cat #4065) following the manufacturer’s protocol. A total of 65 BCs were excluded due to insufficient tissue. TMA slides were first deparaffinized and rehydrated. Epitope retrieval was achieved by boiling the slides for 15 min in citrate buffer pH 6 (Sigma, Cat#C9999) and letting them air-dry before rehydrating and washing them in PBS. The slides were then covered with Dual Endogen Enzyme block (Dako) for 5–10 min at room temperature and washed in PBS (2 × 5 min). After this, anti-human monoclonal mouse IgG₁ S100A8 antibody (R&D systems, Cat #MAB4570, 1:8000) was applied, and the slides were incubated at 4 °C overnight. The next morning the slides were washed in PBS (3 × 5 min) before the secondary antibody and enzyme were added (Dako Labelled polymer-HRP, 30 min at room temperature) followed by another wash in PBS (3 × 5 min). Subsequently, the slides were covered with a mixture of 20 µL DAB chromogen (Dako) and 1 mL Substrate buffer (Dako) for 5–10 min at room temperature, rinsed with water and contrast stained with hematoxylin. Finally, the slides were dehydrated (2 min each, 70%, 85% and 96% ethanol), cleared using xylene, and coverslips were mounted.

After kidney tissues were fixed in 10% formalin, the lumens were inspected for grossly visible lesions. All immunohistochemical studies were performed on paraffin-embedded sections. The paraffin-embedded kidney sections were deparaffinized in xylene, hydrated in graded alcohol and water, and subsequently placed in 3% H₂O₂ to eliminate endogenous peroxidase activity. Next, the sections were blocked with normal goat serum and incubated with the primary antibodies overnight at 4 °C. As a negative control, the primary antibody was replaced with normal rabbit IgG, and staining was performed. All staining methods followed the protocols of Dako Cytomation EnVision + Dual Link System-HRP (DAB+) (Dako Cytomation Inc., Carpenteria, CA). For morphometric analysis, the sections were stained with periodic acid–Schiff (PAS). Masson’s trichrome staining was used to demonstrate collagen deposition. All slides were scanned using an Axiovert 200 M (Zeiss, Jena, Germany) and quantified using NIS-elements BR software 4.0.

Adipose and lung tissue samples were collected after euthanasia (150 mg/kg intraperitoneal, sodium pentobarbital, Thiopentax, Cristalia) at the end of treatment. Tissue samples were fixed in 10% formalin‐phosphate buffer and processed for paraffin inclusion. We obtained sections of 4.5 μm from each tissue using a microtome. The tissue sections were sealed in glass slides and submitted to immunohistochemistry using an anti‐ACE Antibody (2E2: sc‐23908) and anti‐ACE2 antibody (E‐11: sc‐390851) from Santa Cruz and revealed using Dako EnVision® + Dual Link System‐HRP (DAB+). The semi‐quantitative protein expression was evaluated by ImageJ Fiji software (https://imagej.net/Fiji/Downloads).

To identify the origin of primary FM cells obtained from muscular tissue and FISS‐derived cells, ICCs were incubated with vimentin, desmin and anti‐alpha‐smooth muscle actin (α‐SMA) antibodies. The cells were seeded onto a 96‐well plate (4.5 × 10³ cells/well) and incubated at 37°C for 24 h to reach 80%–90% confluence. After removal of the culture medium, the cells in each well were fixed with 80% acetone at −20°C for 10 min, air‐dried at RT and stored at −20°C for subsequent use. One hundred microliters of anti‐vimentin antibody (1:800 dilution) (Dako), anti‐desmin antibody (1:200 dilution) (Dako) and α‐SMA antibody (1:800 dilution) (Dako) were added to each well and incubated for 1 h at RT. After washing with PBS, the EnVision® + Dual Link System‐HRP (DAB+) (Dako) was used following the manufacturer's protocol. The images were evaluated using an inverted microscope (Eclipse TS 100; Nikon) by two pathologists. To quantify the positive cells in the tumour, five high‐power fields were randomly selected and captured and these pictures were analysed using ImageJ software (NIH). The ratio was calculated by dividing the positive cell count by the total cell count. To detect the expression of COX‐2 in FISS and FM cells, ICC was conducted as previously described. The primary antibody was replaced with a COX‐2 polyclonal antibody (Cayman) diluted at 1:200 in PBS.

In total, 44 cases of FTCs and seven cases of HuCCs were subject to immunohistochemical staining. The tissue slides included corresponding normal thyroid tissue for 40 cases. All cases were formalin-fixed paraffin-embedded, cut into 4 μm thick sections and stained using the Envision+ Dual Link System-HRP (DAB+) (DAKO). The tissue slides were incubated with the Anti-Dicer MAB (ab14601, Abcam, diluted 1:100) for 30 min at room temperature before proceeding with the DAKO staining according to the manufacturer’s instructions. The slides were counter-stained with haematoxylin for 5 min and then mounted. The stained slides were subsequently evaluated by an experienced endocrine pathologist and scored as either ‘negative’ (completely devoid of immunoreactivity), ‘weak/focal’ (weak staining in subsets of cells), ‘intermediate’ (diffuse staining of moderate intensity) or ‘strong’ (diffuse staining of strong intensity).