Hrp conjugated secondary antibody

Immunohistochemistry analyses were performed as previously described ^40,41. Dewaxed hydrated paraffin-embedded tissue sections were blocked with 10% normal goat serum in PBS (pH 7.5) and then incubated with primary antibody diluted in 10% normal goat serum in PBS (pH 7.5) overnight at 4°C (anti-ARG1 (1:2000 dilution; ab32037; Abeam). On next day, the sections were incubated with the appropriate species specific HRP-conjugated secondary antibody (2 μg/ml; Vector Laboratories) for one hour at room temperature. Immunoreactivity was detected using the Vectastain Elite DAB kit (Vector Laboratories). A semiquantitative grading system (H-score) was used to compare the immunohistochemical staining intensities as previously described ⁴². The overall score ranged from 0 to 300.

Patients with HCC are summarized in Supplementary Table S2. Formalin-fixed paraffin-embedded tissue specimens were cut into 4 μm sections. After dewaxing in xylene, tissue sections were autoclaved for 10 min in a citrate buffer, pre-incubated in 0.3% H₂O₂, and blocked for 20 min using diluted normal blocking serum from a VECTASTAIN Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA). Sections were incubated with 1:100 diluted anti-URI1 antibody as a primary antibody followed by an HRP-conjugated secondary antibody (Vector Laboratories). URI1 protein was visualized using ImmPACT DAB substrate (Vector Laboratories) with a reaction time of 3 min.
Paired HCC-B and non-tumor liver tissues from five patients (Supplementary Table S3) were used for the ChIP assay, which was performed as previously reported [41 (link)].
These studies were approved by the ethical committee of Tottori University (18A071).

KDR protein was identified by immunocytochemistry in HUVEC isolated from normal pregnancies and HUVEC exposed to conditioned medium (CM) from GDM HUVEC (see above) by using a commercial kit (Vector Laboratories, MI, USA) following the manufacturer’s protocol. Briefly, after treatment HUVECs were fixed in 4% paraformaldehyde prepared in phosphate buffer (PBS, (mM): NaCl 13.7, KCl 2.7, Na₂HPO₄ 0.9, KH₂PO₄ 1.8, pH 7.4, 4°C) for 20 minutes. After blocking unspecific binding using 5% bovine serum albumin (BSA) in blocking buffer solution (PBS/Tween 1%), the cells were incubated overnight with primary KDR antibody (Cell Signaling Technology, Boston, USA) (Clone 55B11, dilution 1:200 v/v). Antigen-antibody reaction was further revealed by HRP-conjugated secondary antibody (Vector Laboratories, MI, USA).
Images were taken by a camera and the intensity of the immunocytochemical staining was semi-quantified using ImageJ software (National Institutes of Health, Bethesda, MD) [24 (link)]. Values are expressed as the ratio between the area of brown stain and the total area of the reference field. All samples for digital and microscopic analyses were blinded to the operator.

Uterine sections from paraffin-embedded tissues were cut at 5 μm and mounted on silane-coated slides, deparaffinized, and rehydrated in a graded alcohol series before blocking with 10% normal goat serum in PBS (pH 7.5) and incubating with primary antibody diluted in 10% normal goat serum in PBS (pH 7.5) overnight at 4°C at the following dilutions: 1:500 for anti-ARID1A (Sc-98441, SantaCruz), 1:100 for anti-Ki67 (ab15580, Abcam), anti-ESR1 (DAKO Corp.), 1:100 for anti-phospho-ESR1 (Ab31477, Abcam), 1:1000 for MUC-1 (ab15481, Abcam), 1:2000 for LTF (07–682, Millipore), MA), 1:20000 for MCM2 (Sc-9839, SantaCruz), 1:20000 for MCM6 (Sc-9843, SantaCruz), 1:5000 for KLF4 (Sc-20691, SantaCruz), 1:5000 for FLK15 (ab2647, Abcam), and 1:1000 for anti-total PGR antibody (A0098, DAKO Corp.). On the following day, sections were washed in PBS and incubated with the appropriate species-specific HRP-conjugated secondary antibody (2 μg/ml; Vector Laboratories) for 1 hr at room temperature. Immunoreactivity was detected using the Vectastain Elite DAB kit (Vector Laboratories). A semiquantitative grading system (H-score) was used to compare the immunohistochemical staining intensities as previously described [101 (link)]. The number of PGR and Ki67-positive cells was counted in 200 epithelial cells and eight random fields of stromal cells.