Diaminobenzidine dab substrate

Mouse mammary tissues were extracted, fixed in formalin and embedded in paraffin before sectioning at 5 μm. Haemotoxylin and eosin staining was carried out according to standard protocols. For immunohistochemical analysis of Ki67, slides were immersed in 0.5 % hydrogen peroxide in methanol for 10 minutes to inhibit endogenous peroxidase activity, followed by antigen retrieval by boiling slides in 0.1 M sodium citrate buffer under pressure. Slides were blocked for 20 minutes in 5 % normal rabbit serum in TBS/0.1 % Tween to prevent non-specific antibody binding, and then incubated overnight at 4 °C with mouse anti-Ki67 antibody (Vector Labs) according to the manufacturer’s instructions. Specific antibody binding was detected using the EnVision Dual Link System (Vector Labs), followed by incubation with diaminobenzidine (DAB) substrate (Dako). Sections were counterstained with haemotoxylin, dehydrated and mounted.

Tumors tissues were fixed in 4% PFA, dehydrated and embedded in paraffin. The paraffin-embedded tissue blocks were cut into 5 μm-thick sections and dewaxed, rehydrated, blocked and then incubated with individual primary antibody at room temperature for 60 min. Primary antibodies used in this study were Ki-67 (Abcam: ab16667), CD31 (Abcam: ab9498), E-cadherin (CST: #3195), N-cadherin (CST: #13116) and Vimentin (CST: #5741). Positive signals were developed using diaminobenzidine (DAB) substrate (Dako Carpinteria, CA, USA) under the manufacturer recommended conditions and photographed^{12 (link)}. Experiment and result assessment were conducted blind.

Paraffin-embedded tissues were used to conduct IHC staining. Section slides with tissue were deparaffinized in xylene and rehydrated via graded ethanol (dilution from 100%, 95%, 85% and 75%). Blockage of endogenous peroxidase activity and antigen retrieval was performed before incubation with primary antibody at 4 °C overnight. Staining was then conducted with diaminobenzidine (DAB) substrate (Dako) after incubation with secondary antibody (HRP-conjugated) for 20 min at room temperature. Hematoxylin was used to stain sections after DAB treatment. The antibodies used are listed in Supplementary Table 6. The staining intensity of each section was scored as 0 (no staining), 1+ (weak staining), 2+ (moderate staining), or 3+ (strong staining), and the percentage of positive cells was determined from five different random regions. H-score method (score range, 0–300) was used to quantify the expression. H-score from 0 to 200 was considered as low expression and H-score from 201 to 300 was assigned as high expression. For H&E staining, the sections were immersed in hematoxylin for 3 min, washed with water for 30 min, and dyed with eosin for 3 min. The slides were covered with coverslips after dehydration in ethanol at different concentrations. The stained slides were imaged under an optical microscope (NIKON ECLIPSE 80i).

Section slides with tissue were deparaffinized in xylene and infiltrated with graded concentrations of ethanol (100%, 95%, 85%, and 75%). After performing the blocking of endogenous peroxidase activity and antigen retrieval, the Ki-67 (Proteintech, China, 1:200) antibody was added and incubated overnight at 4° C. The sections were stained with a diaminobenzidine (DAB) substrate (Dako) after 20 minutes of incubation at room temperature. The sections were stained with hematoxylin after DAB treatment. The sections slides were dried, and the degree of staining and the positive range were measured under a microscope. The degree of staining (0-3 points) was negative, light yellow, light brown, and dark brown; the positive range (1-4 points) was respectively 0-25%, 26-50%, 51-75%, 76-100%, and the final scores were added for comparison. And we have added the related content to the Methods section according to the degree of staining and the positive range under a light microscope.

Tumor and adjacent mammary tissues of TNBC patients utilized for IHC were retrospectively obtained from the Sun Yat‐Sen University Cancer Center, with prior informed consent obtained from each patient. Tissue‐containing sections were deparaffinized with xylene and subsequently rehydrated using a series of graded ethanol dilutions (100%, 95%, 85%, and 75%). Prior to overnight incubation with the primary antibody at 4°C, endogenous peroxidase activity was blocked and antigen retrieval was conducted. Subsequent to a 20‐min incubation with an HRP‐conjugated secondary antibody at ambient temperature, staining was performed utilizing diaminobenzidine (DAB) substrate (Dako). Following DAB treatment, sections were stained with hematoxylin.

Tissues were fixed in formalin and embedded in paraffin, then sections (4 µm) were cut. Following deparaffinization with xylene, the sections were rehydrated with graded ethanol and washed with PBS, then stained with hematoxylin and eosin for histological analysis. The immunohistochemistry staining of sections was performed by using the Dako EnVision FLEX kit (Dako, Glostrup, Denmark) according to the manufacturer’s instructions as previously described [44 (link)]. Briefly, the sections were subjected to antigen retrieval in Target Retrieval Solution (Dako, Glostrup, Denmark). Endogenous peroxidase was blocked with Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark). Then, the sections were incubated with primary antibody at 4 °C overnight and followed by incubation with secondary antibody FLEX/HRP (Dako, Glostrup, Denmark) at room temperatures for 30 min. Staining was developed by diaminobenzidine (DAB) substrate (Dako, Glostrup, Denmark). The stained sections were scanned by Pannoramic MIDI (3D HISTECH, Budapest, Hungary). Primary antibodies used in this study were Ki67 (Abcam, Cat# ab16667), PCNA (CST, Danvers, MA, USA, Cat# 13110), Cyclin D1 (CST, Danvers, MA, USA, Cat# 55506), E-Cadherin (CST, Danvers, MA, USA, Cat# 3195), and N-Cadherin (CST, Danvers, MA, USA, Cat# 13116).