Streptavidin biotin peroxidase complex

Multiplex immunohistochemistry/immunocytochemistry (mIHC/mICC) was performed as previously described [56 (link)]. Paraffin-embedded luteal sections were first dewaxed and subjected to microwave treatment for antigen retrieval. The luteal cells were seeded on glass coverslips (Solarbio, Beijing, China). The endogenous peroxidase activity was quenched by hydrogen peroxide. Slides were then blocked and incubated with primary antibody (Table 1, APN, T-Ca, AdipoR1, AdipoR2, P-AMPK, AMPK, HSD3B, STAR) at 4 °C overnight. The negative controls (NC) were established by replacing the primary antibody with normal serum [57 (link)]. Sections were further incubated with a standard streptavidin–biotin–peroxidase complex (Boster Biological Technology, Wuhan, China) at room temperature. Then, a chromogenic working solution was added and optimized by employing a triplex (AF488, Cy3 and Cy5, ATT, Dallas, TX, USA) and were finished with DAPI. Image capture was performed with a Zeiss LSM900 (Zeiss, Jena, Germany).

For in situ hybridization (ISH), CRC, and adjacent normal tissue specimens were fixed with 4% para-formaldehyde, dehydrated, and embedded in paraffin. The specimens were sliced into 4-μm-thick sections and mounted onto charged slides. After dewaxing and hydration, the sections were air-dried and immersed in distilled water containing 3% hydrogen peroxide, followed by immersion in pepsin solution for 30 min at 37°C. The sections were treated with hybridization buffer for 2 h at 37°C. Next, hybridization was performed by incubating the sections with the target DIG-labeled probe (AAGAAGCTGCTGAAGAACCCA) at 42°C overnight, followed by washing with 2 ×, 0.5 ×, and 0.2 × SSC solution, respectively. The sections were then blocked with biotinylated digoxin (Boster Biological Technology, Wuhan) for 1 h at 37°C and the streptavidin-biotin-peroxidase complex (Boster) for 20 min at 37°C. Hybridization signals were detected using DAB (3,3′-diaminobenzidine; P013IH, Auragene), and the sections were counterstained with hematoxylin. After dehydration, the sections were mounted with neutral gum and observed under the microscope.

Coronary vascular tissues were fixed, sectioned, and sealed. After that, tissues were reacted with the primary antibody vascular endothelial growth factor (VEGF, 1:100, sc-7269, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and with goat anti-rabbit secondary antibody immunoglobulin G (IgG, ab150077, 1:200, Abcam, MA, USA) in phosphate-buffered saline (PBS). Then, the tissues were kept in streptavidin biotin-peroxidase complex (Boster Biological Technology Co. Ltd., Hubei, China), stained with DAB (Boster), and observed by a microscope (Leica) [18 (link)].
Immunohistochemistry was applied to determine vWF in ECs. The collected cells were placed in 4% paraformaldehyde, frozen in 30% sucrose PBS, and sectioned into 30 μm by a cryostat. Subsequently, cells were reacted with 0.3% H₂O₂ or methanol and sealed with 10% PBS. With En Vision two-step method, cells were probed with rabbit anti-human factor VII-related antigen polyclonal antibody (Neomarkers, CA, USA) and with anti-rabbit/mouse universal immunohistochemical kit (DAKO, MI, USA). Followed by that, cells were developed by DAB, counterstained by hematoxylin solution, treated with ammonia, and observed under an optical microscope (Olympus).