Tumor sections was obtained from post-surgery specimens with an informed consent waiver. Postoperative tumor tissues derived from patients were fixed in 4% paraformaldehyde and embedded in paraffin. The tissues were sectioned into 5-μm slices and deparaffinized with xylene followed by rehydrated with graded alcohols. Antigen retrieval was performed in a boiling pressure cooker with citrate buffer (pH = 6.0) for 10 min. The sections were incubated with anti-ESM1 antibody (bs-3615R, Bioss, Beijing, China) at a dilution of 1:200 overnight at 4°C. After washing with PBST the next day, the sections were incubated with an HRP-conjugated anti-rabbit secondary antibody (MaxVision, Fuzhou, China) for half an hour at room temperature. Finally, the sections were visualized with diaminobenzidine (DAB, ZSGB Bio, Beijing, China) and counterstained with hematoxylin. The IHC images were captured by a slide Scanner System (3DHISTECH, Budapest, Hungary) and Immunohistochemistry scores (H-scores) were quantified by 3DHISTECH QuantCenter software (3DHISTECH, Budapest, Hungary). Since there is no universally accepted standard, we considered a score below 50 as negative expression, 50-100 as weak positive, 100-150 as medium positive, and greater than 150 as strong positive based on previous literature (14 (link)).
Diaminobenzidine dab
Manufactured by ZSGB-BIO
Sourced in China
Diaminobenzidine (DAB) is a chromogenic substrate used in immunohistochemistry and immunocytochemistry applications. It is a sensitive, stable, and insoluble reaction product that results in a brown staining when oxidized by peroxidase enzymes. DAB is commonly used to visualize the presence and localization of target proteins or antigens in tissue sections or cell cultures.
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Lab products found in correlation
Image pro plus software, by Media Cybernetics (2 mentions)
Ab16667, by Abcam (2 mentions)
Bs 3615r, by Bioss Antibodies (1 mentions)
Slide scanner system, by 3DHISTECH (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Prussian blue staining kits, by Leagene (1 mentions)
Collagenase 7, by Merck Group (1 mentions)
Antibodies against ki 67, by Abcam (1 mentions)
Goat anti rabbit secondary antibody, by Boster Bio (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit igg, by Bioss Antibodies (1 mentions)
Hematoxylin, by Nanjing Jiancheng (1 mentions)
Splink detection kit, by ZSGB-BIO (1 mentions)
Bdnf antibody, by Abcam (1 mentions)
Magnification light microscope, by Olympus (1 mentions)
Edta buffer, by ZSGB-BIO (1 mentions)
Ab5831, by Abcam (1 mentions)
22 protocols using diaminobenzidine dab
1
Immunohistochemical Analysis of ESM1 Expression
2
Immunohistochemical Analysis of HSP60 Expression
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Tissue sections were mounted on slides, treated with xylene, and rehydrated using a graded series of alcohol–water solutions before heat treatment in 10 mM citric acid (pH 6.0) for antigen retrieval49 (link). Endogenous peroxidase activity was quenched using a 0.3% H2O2 solution. After blocking with 10% serum, sections were incubated at 4 °C overnight with primary anti-HSP60 antibodies (sc-376261; 1:200; Santa Cruz Biotechnology, Dallas, TX, USA). The slides were then incubated with anti-mouse IgG antibody (ZSBIO, Beijing, China) at 37 °C for 30 min. Signals were visualized with diaminobenzidine (DAB) (ZSBIO), counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and mounted. Protein expression levels were analyzed by calculating the integrated optical density per stained area (IOD/area, mean OD value, MOD) using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) as described previously50 (link).
3
Histochemical Analysis of Cells
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Collagenase VII and 4′,6‐diamidino‐2‐phenylindole (DAPI) were from Sigma. Collagenase VII was dissolved in physiological saline solution at concentration of 25 U/μL and stored at −20°C. Before use, Collagenase VII was diluted to 0.2 U/μL in 0.9% NaCl. All the antibodies used in this study are listed in Table S1 . Prussian blue staining kits were from Leagene; Histostain TM‐SP kits were from ZEMED; and diaminobenzidine (DAB) was from ZSGB‐Bio. Emodin was obtained from Shanghai Base Industry and dissolved in DD water (0.6%). Lactate was obtained from Sigma.
4
Immunohistochemical Analysis of Ki-67 Expression
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Immunohistochemistry was performed to detect Ki-67 expression in the mouse tumor tissues. The antibodies against Ki-67was from Abcam, and the peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) and diaminobenzidine (DAB) were from ZSGB-BIO (Beijing, China).
5
Immunohistochemical Staining Protocol
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Prepared paraffin-embedded sections were deparaffinized, rehydrated, retrieved and blocked with 5% BSA (w/v) in PBS at ambient temperature for 30 min. Then, sections were incubated with 100 μM biotin labeled RP-1 peptide or human specific rabbit anti-CD44 monoclonal antibody (1:200, ZSGB-BIO, Beijing, China) overnight at 4°C. After PBS washing for three times, sections were subsequently incubated with streptavidin-HRP (1:500, Proteintech, Wuhan, China) and goat anti-rabbit secondary antibody (1:300, BOSTER, Wuhan, China). Diaminobenzidine (DAB, ZSGB-BIO, Beijing, China) was used as a chromogen. Finally, the sections were counterstained with hematoxylin and mounted with neutral balsam.
6
Immunohistochemical Analysis of ST6Gal-I in HCC
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ST6Gal-I levels in hepatoma carcinoma and normal hepatic tissues were evaluated by IHC using anti-ST6Gal-I on commercial tissue arrays (Shanghai Zhuo Li Biological Technology, China) as previously described.38 (link), 39 (link) After antigen retrieval and blocking, the slides were incubated with a rabbit anti-ST6Gal-I primary antibody (1:100; Abcam, Cambridge, UK) overnight at 4 °C. Negative control slides were processed in the same manner, without addition of the primary antibody. For detection, diaminobenzidine (DAB) (ZSGB-BIO, Beijing, China) and hematoxylin were used. Two certified pathologists blinded to the clinical data independently assessed ST6Gal-I immunostaining, scoring the results depending on the positive-staining percentages and staining intensity, as follows: (0) 0%, no staining (–); (1) 1–29%, weak positive (+); (2) 30–59%, moderate positive (++); and (3) >60%, intense staining (+++). For statistical analysis, cases were categorized as either negative (no and weak staining) or positive (moderate and intense staining). Pearson’s chi-squared test was used to assess the association between ST6Gal-I and HCC, to determine its clinical significance.
7
Immunohistochemical Analysis of Ki-67 Expression
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Sections of transplanted tumour tissues were obtained, and endogenous peroxidase was blocked with 30% H2O2. An antigen repair solution was added to the sections before boiling. After cooling for 5 min, the processes of boiling and cooling were repeated two times. After cooling at room temperature, the sections were incubated with 5% BSA at room temperature for 20 min, followed by the removal of the excess liquid. Subsequently, the sections were incubated with rabbit anti‐mouse Ki‐67 primary antibody (ab15580, 1:1000; Abcam, Cambridge, MA, USA) at 4°C overnight. The sections were incubated in biotinylated sheep anti‐rabbit IgG (ab6721; Abcam, Cambridge, MA, USA) at 37°C for 40 min. After washing with PBS, the sections were developed with diaminobenzidine (DAB; ZSGB‐Bio, Beijing, China). The Multifunctional True Color Cell Image Analysis and Management system (Media Cybernetics, Rockville, MD, USA) was applied for analysis. Three sections were selected from each sample, and three fields were selected from each section. Quantitative analysis of the images was performed with the Image‐pro Plus Software (Media Cybernetics, Rockville, MD, USA). The integral optical density (IOD) of the Ki‐67 positive staining was measured, and the IOD value was used to represent the expression of Ki‐67.
8
Comprehensive Western Blot Analysis of Cell Cycle and Stress Signaling
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Western blotting was performed as previously described (36 (link)). Equal amounts of protein per sample were separated by 12% SDS-PAGE and transferred to the PVDF membranes (Thermo Fisher Scientific, Waltham, MA, USA). Protein bands were visualized with an enhanced chemiluminescence assay kit (Syngene, Cambridge, UK), and signal intensity of protein bands was analyzed by the use of Image J software. The immunoblots were incubated with primary antibodies against cyclin A (1:1000 dilution in PBST), cyclin B (1:1000), cyclin D (1:500), cyclin E (1:750), CDK1 (1:500), CDK2 (1:1500), CDK4 (1:500), CDK6 (1:500), E2F1 (1:500), E2F2 (1:1000), E2F3 (1:500), E2F4 (1:1000), RB (1:500), p21 (1:1000), p27 (1:1000), p53 (1:500), GRP78 (1:2000), IRE1 (1:500), p-IRE1 (1:500), XBP1s (1:1000), PERK (1:1000), p-PERK (1:500), ATF4 (1:1000), CHOP (1:500), ATF6 (1:500), Skp2 (1:500), pro-CASP3/cl-CASP3 (1:500), pro-PARP/cl-PARP (1:500), AKT (1:500), p-AKT (1:500), JNK (1:500), p-JNK (1:500) and β-actin (1:8000) probed with the secondary antibody HRP conjugated goat anti-rabbit IgG (1:5000; Bioss, Beijing, China), and visualized by diaminobenzidine (DAB) (ZSGB-BIO, Beijing, China). Primary antibodies were obtained from two commercial sources (ABclonal, Wuhan, China; Bioss, Beijing, China).
9
Histological and Immunohistochemical Analysis of Liver Tissue
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Liver tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Cross sections (5 µm) of the tissue were cut and then mounted on glass for histology and immunohistochemical analysis. hematoxylin and eosin (H&E) staining was performed to assess liver tissue morphology. Immunohistochemical staining was described as before (Lv et al., 2019 (link)). In brief, the liver tissues were embedded in paraffin and sliced into 5-μm sections. After deparaffinization and rehydration, the sections were incubated in sodium citrate buffer. Then 1% H2O2 was used to suppress the endogenous peroxidase activity. Thereafter, the sections were incubated with an anti-CD68 antibody at 4°C overnight. The sections were then incubated with a secondary antibody—anti-rabbit IgG (Santa Cruz, CA, United States) for 1 h at room temperature. Finally, immunospecific reactivity was visualized by diaminobenzidine (DAB; ZSGB-BIO, Beijing, China) substrate and counterstained with hematoxylin (Jiancheng Biotech, Nanjing, China). Images were acquired with a microscope (Leica, Solms, German).
10
Immunohistochemical Detection of PRV Antigen
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The paraffin-embedded tissue samples were sectioned to 4 μm and stained with hematoxylin-eosin (HE). For immunohistochemical studies, the sections were examined for the presence of PRV antigen with a horseradish peroxidase (HRP) method using a primary HRP-conjugated anti-PRV mouse monoclonal antibody (provided by Prof. Hanchun Yang, Key Laboratory of Animal Epidemiology and Zoonosis, China Agricultural University, China). The antigen–antibody complexes were examined with the Polink-2 Plus HRP Detection Kit (GB-BIO, Beijing, China), according to the manufacturer’s instructions. The sections were incubated with diaminobenzidine (DAB; ZSGB-BIO, Beijing, China) for visualization. Finally, the slides were counterstained with hematoxylin. Sections of brainstem from a naturally PRV-infected dog were used as the positive control for each series of stained sections. Sections of brainstem from a healthy dog were included as the negative control.
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