Dab horseradish peroxidase color development kit

Heart samples were collected and fixed overnight in 4% paraformaldehyde (BL539A, Biosharp, Hefei, Anhui, China), followed by routine dehydration and sectioning (5 μm). Sections were blocked with 3% hydrogen peroxide, antigen repaired with citrate buffer (P0083, Beyotime, Shanghai, China), permeabilized with 0.3% Triton-100 (ST795, Beyotime, Shanghai, China), blocked with 5% BSA (A1933, Sigma-Aldrich, St. Louis, MO), incubated with primary antibody overnight at 4 °C and incubated with HRP-labeled secondary antibody for 30 min at 37 °C. Visualization was performed under the microscope (DFC700T, Leica, Germany) with DAB Horseradish Peroxidase Color Development Kit (P0203, Beyotime, Shanghai, China). Finally, the sections were sealed with neutral balsam fixative (G8590, Solarbio, Beijing, China). The positive cell number was counted from 4–5 fields per sample with the ImageJ software program (v.1.45, National Institutes of Health, Bethesda, MD, USA) and the mean density was quantified from 4–5 fields per sample with the Image-pro plus software program (v.6.0, Media Cybernetics, Rockville, MD, USA). The antibodies are listed in Supplementary Table 1.

Testicular samples derived from testicular biopsy were fixed with 4% paraformaldehyde and embedded with paraffin. For histological analysis, testicular sections were prepared and stained with hematoxylin and eosin. For the detection of PIWIL2 protein, the testicular sections were prepared and blocked with 5% bovine serum albumin (BSA), followed by incubation with PIWIL2 or isotype (all from Abcam, UK) primary antibody (Additional file 4: Table S2) at 4 °C overnight. After incubation with a secondary antibody (Additional file 4: Table S2) at room temperature for 1 h, the sections were visualized with the use of the DAB Horseradish Peroxidase Color Development Kit (Beyotime, China). Nuclei were stained with hematoxylin. The sections were then observed under an Olympus microscope.

Tumor tissues of three randomly chosen mice per group were fixed in 4% formaldehyde, embedded in paraffin, and sectioned. Immunostaining was carried out with rabbit primary antibodies (Abcam, Inc., Shanghai, China) against three different interleukins, i.e., IL-27-A, IL-5RA, and IL-9, respectively. The secondary antibody was a goat anti-rabbit horseradish peroxidase-linked immunoglobulin (Cell Signaling Technology, Inc., Shanghai, China). The DAB Horseradish Peroxidase Color Development Kit (Beyotime, Beijing, China) was used for reacting with the sections for color development. Stained sections were imaged with Leica Aperio CS2 system (Leica, Wetzlar, Germany). Positive signals of immunostaining of histological sections were quantified by using ImageJ (three mice per group, two replicates per mouse per staining). Data are presented as average optical density.

To further observe the morphological structure and growth of tumor in each group, HE staining, Caspase-3 and FANCG immunohistochemical staining were used to detect tumor in each group [17 (link), 18 (link)]. The mice were sacrificed at the end of the experiment and the tumor tissues were fixed in 10% formalin, paraffinized, and cut into 5 μm thick sections.

HE staining.

The slides were dewaxed and stained with hematoxylin–eosin for histological assessments.
(b)

Immunohistochemistry.

After microwave pre-treatment in a citrate buffer (pH 6.0; for antigen retrieval), the slides were immersed in 3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. After intensive washing with PBS, the slides were incubated with Caspase-3, FANCG antibodies and then incubated with HRP-conjugated antibodies. The slides were visualized with a DAB Horseradish Peroxidase Color Development Kit (Beyotime, China), and counterstained with hematoxylin. The images were analyzed by Image-Pro Plus 6.0 software.

Human samples were obtained as described previously [17 (link)]. Briefly, a trauma patient required popliteal artery and vein reconstruction with a spiral saphenous vein graft (SVG) in the popliteal vein, amputation was performed on day 18 after vascular reconstruction because of the patient’s serious injuries, and all protocols involving human biospecimens complied with all relevant ethical regulations. Tissues were processed and stained as described previously, briefly, the SVG was fixed and embedded in paraffin and sectioned (4 μm thickness); sections were heated in citric acid buffer (pH 6.0) for antigen retrieval and then treated with 0.3% hydrogen for 30 min, then the sections were incubated with PD-1 or TGF β1 antibody; after overnight incubation, the sections were incubated with appropriate secondary antibodies for 1 hr at room temperature and treated with DAB Horseradish Peroxidase Color Development Kit (Beyotime, Shanghai, China) to detect the reaction products; finally, the sections were counterstained with hematoxylin (BASO) [17 (link)].