Ab238697

During the colposcopy, cervical samples were taken from the areas that were evaluated as abnormal with punch biopsy forceps. All cervical specimens were fixed with formalin and embedded in paraffin. Sections that were 4-μm thick were cut and stained with hematoxylin and eosin. For the histological examination, we evaluated the presence of cell dysplasia, cell irregularity, cell hyperchromatism, and increased nuclear-cytoplasmic ratio, loss of cell polarization, mitotic figure, koilocytosis, and infiltration of the immune cells.
All tissue preparation and immunohistochemistry (IHC) procedures were performed as previously described [17 (link)]. IHC analysis was performed using the Super Vision Assay Kit (SV0002–1, Boster Bio, Pleasanton, CA, USA) by two experienced histopathologists. The primary antibodies used in this study were an anti-human Ki-67 antibody against proliferating cells, dilution 1:100 (Anti-human Ki-67 Antigen Antibody, Dako, Carpinteria, CA, USA), an anti-mouse p16 (p16INK4a) monoclonal antibody, dilution 1:50 (Roche E6H4™, catalog #725–4713, Roche, Basel, Switzerland), and an anti-mouse Anti-PD-L1 antibody, dilution 1:100 [PDL1/2746] (ab238697, Abcam, Cambridge, MA, USA). During the evaluation, analyzed fields were randomly selected from ten slides using a light microscope (Nikon Eclipse Ni-U, Tokyo, Japan).

Cells were collected and lysed in RIPA buffer containing protease inhibitor cocktail. After quantification, 20 μg of total proteins were used to perform an SDS-PAGE and WB analysis. PVDF membranes were incubated overnight with the primary antibodies anti-PD-L1 (ab238697, Abcam), phospho-CREB (ab32096, Abcam), CREB (ab32515, Abcam), p-PKAα/β/γ (sc-32968, Santa Cruz Biotechnology), β-actin (sc-1615, Santa Cruz Biotechnology), β3-AR (ab94506, Abcam) at 4 °C and then with specific secondary antibodies for 1 h at room temperature. Binding of the antibodies with the specific proteins has been detected by using Clarity Western ECL Substrate (Bio-Rad) and images were acquired through the Chemidoc Imaging System (Biorad®). IMAGEJ software was used to perform quantitative analyses, and the signal intensity of the bands was expressed as fold-increase relative to control values.

Immunofluorescence analysis of murine tumor mass was performed as follows. Samples were rapidly excised, fixed in buffered 4% formaldehyde for 24 h and paraffin-embedded. Histological sections, 5 μm thick, were cut from samples, were deparaffinized and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0; Bio-Optica) for antigen retrieval and immunostained over night at 4 °C with primary antibodies (anti-PD-L1, ab238697, Abcam; anti-β3-AR, ab94506, Abcam; anti-CD4, 14-9766-82, eBioscience; anti-CD8, 14-0808-82, eBioscience; DBH, ab209487, Abcam). Immune reaction was revealed incubating sections with secondary antibodies Alexa Fluor 488-conjugated IgG or Alexa Fluor 594-conjugated IgG (1:350; Jackson Laboratory). After counterstaining with 4,6-diamidino-2-phenylindole (DAPI), representative images were acquired by an Olympus BX63 microscope coupled to CellSens Dimension Imaging Software version 1.6 (Olympus). The immunofluorescence staining was evaluated through the ImageJ software (NIH, USA) as the fluorescence intensity of protein of interest was normalized to DAPI values.

Formalin-fixed tumours were embedded in paraffin, sectioned at 4 μm and mounted on SuperFrost ULTRA PLUS slides (Thermo Fisher Scientific). Sections were deparaffinized, rehydrated in a series of alcohols, and microwaved in citrate buffer pH = 6 for heat-induced epitope retrieval. Sections were blocked and stained with the following antibodies: anti-human PD-L1 antibody (#ab205921, Abcam) or anti-mouse PD-L1 (#ab238697, Abcam). Primary antibodies were detected using the EnVision + System-HRP labelled Polymer and Liquid DAB + substrate chromogen system (Agilent Technologies). All procedures were performed at room temperature and all tumours were stained in the same batch.