After routine deparaffinization and hydration, tissue sections were treated with 0.01 M citrate buffer (pH 6.0) for antigen retrieval. Following 3% hydrogen peroxide incubation and 10% bovine serum albumin deprivation, slides were incubated with primary antibodies of c-Fos (1:100, Cell Signaling), Wnt2 (1:100, Abcam), and Fzd9 (1:100, Abcam) at 4°C overnight. Primary antibodies were recognized by the biotinylated secondary antibody and visualized by Vectastain avidin–biotin complex peroxidase system (Zsbio, China) and 3,3′-diaminobenzidine kit (DAB, Zsbio, China). The degrees of immunoreactivity were evaluated in accordance with our previous report[14 (link)]. We performed staining by only adding phosphate-buffered saline (PBS) instead of using primary antibodies to the sections as negative controls. The sections were photographed using an optical microscope (Olympus, BX53F, Japan) and then analyzed using the Image-Pro Plus 6.0 analytic system.
3 3 diaminobenzidine kit
Manufactured by ZSGB-BIO
Sourced in China
The 3,3′-diaminobenzidine kit is a laboratory product used for the detection and visualization of specific target molecules or proteins in biological samples. It provides a chromogenic reaction that produces a brown precipitate, enabling the identification and localization of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Sp9000 polymer detection system, by ZSGB-BIO (7 mentions)
Apopcyto caspase 3 colorimetric assay kit, by Medical & Biological Laboratories (2 mentions)
Image pro plus 6.0 analytic, by Olympus (1 mentions)
C fos, by Cell Signaling Technology (1 mentions)
Bx53f, by Olympus (1 mentions)
Sp 9001, by ZSGB-BIO (1 mentions)
Ab242370, by Abcam (1 mentions)
Ab272692, by Abcam (1 mentions)
Ab221547, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Goat anti rabbit igg, by ZSGB-BIO (1 mentions)
Primary antibody against, by Santa Cruz Biotechnology (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
13 protocols using 3 3 diaminobenzidine kit
1
Immunohistochemical Analysis of Wnt Signaling
2
Immunohistochemical Scoring Protocol
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Paraffin-embedded tissue sections were baked for 3 h at 60 °C and subjected to microwave treatment in citrate buffer for antigen retrieval. Next, the samples were incubated with 3% H2O2 for 15 min and blocked with 3% bovine serum albumin (BSA) for 1 h. The sections were incubated with primary antibodies overnight at 4 °C, followed incubated with the secondary antibodies (ZSGB-Bio, sp9001) for 30 min. Immunoreactive signals were developed using a 3,3′-diaminobenzidine kit (ZSGB-Bio). The sections were counterstained with hematoxylin.
The immunoreactive signal intensity was scored as follows: 0, negative staining; 1, weakly positive staining (light brown); 2, moderately positive staining (brown); and 3, strongly positive staining (dark brown). Additionally, the immunoreactive signals were quantified as follows: 0, negative; 1, positive cells ≤ 25%; 2, 26-50% positive cells; 3, 51-75% positive cells; and 4, positive cells > 75%. The EI value, which ranged from 0 to 12, was determined by multiplying the extent (E) and intensity (I) scores.
The immunoreactive signal intensity was scored as follows: 0, negative staining; 1, weakly positive staining (light brown); 2, moderately positive staining (brown); and 3, strongly positive staining (dark brown). Additionally, the immunoreactive signals were quantified as follows: 0, negative; 1, positive cells ≤ 25%; 2, 26-50% positive cells; 3, 51-75% positive cells; and 4, positive cells > 75%. The EI value, which ranged from 0 to 12, was determined by multiplying the extent (E) and intensity (I) scores.
3
Immunohistochemical and Histopathological Analysis of Mouse Intestine
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The small intestinal tissue of mice was fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into sections at a thickness of 5 μm (The histopathology associated index of intestine was evaluated though the criterion in Supplementary Table 1 ). For immunohistochemical staining, the intestinal sections were subjected to deparaffinization, hydration, antigen retrieval, quenching of endogenous peroxidase, and blocking procedures. All slices were then incubated with the primary antibodies against pSTAT3 (CST #9145) and Ki-67 (Abcam, England, Ab16667) at 4°C overnight followed by incubation with biotinylated secondary antibodies for 30 min and visualization using a 3,3′-Diaminobenzidine Kit (ZSGB-BIO, Beijing, China). For immunofluorescence staining for the detection of MUC2 (Abcam, England, Ab272692), Lgr5 (NBP1-28904SS), ZO-1 (Abcam, England, Ab221547), and Claudin-1 (Abcam, England, Ab242370), the colonic sections were processed by the methods described above. Periodic Acid-Schiff stain (PAS) staining (Biossci, China) was performed following the manufacturer’s protocols.
4
Immunohistochemical Analysis of AFP and CXCR4
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The expression and cellular distribution of AFP and CXCR4 protein were assessed by immunohistochemical analysis. Five-millimeter-thick paraffin sections were deparaffinized and re-hydrated according to standard protocols, and heat-induced antigen retrieval was performed in sodium citrate buffer (10mmol/L, pH 6.0). Endogenous peroxidase was inhibited by 0.3% H2O2, and non-specific protein binding was blocked with 10% goat serum. The sections were then incubated with primary antibody against AFP and CXCR4 (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. Non-immune immunoglobulin G(IgG) was used as a negative control, and antigenic sites were localized using a SP9000 Polymer Detection System and a 3,3′-diaminobenzidine kit (ZSGB-BIO, Beijing, China).
5
Immunohistochemical Analysis of Kidney Tissue
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Human kidney specimens and mice kidney sections were fixed with 4% paraformaldehyde (PFA) for over 24 h and then were embedded in wax before being sliced into 3‐µm sections. After dewaxed gradually with xylene and ethanol, the tissue slides were microwaved in citrate antigen repair solution (Cat #P0083, Beyotime Biotechnology, Shanghai, China) for 10 min and then treated with 3% H2O2 for 10 min to block the endogenous peroxidase activity. Subsequently, the tissue slides were blocked (Cat #P0260, Beyotime Biotechnology) for 1 h at room temperature and then incubated with primary antibodies: anti‐TP53RK (Cat #ab279377, Abcam, Cambridge, MA, USA) and anti‐Birc5 (Cat #ab469, Abcam) overnight at 4 °C. The next day, horseradish peroxidase was applied. Localization of peroxidase conjugates was determined using a 3, 3'‐diaminobenzidine kit (Cat #PV‐9000, Zsbio, Beijing, China) and nuclei were stained with hematoxylin. Images of tissue slides were collected with an Olympus BX51 microscope (Olympus, Tokyo, Japan) and the intensity of the positive staining area was quantified with the Image‐Pro Plus software (Media Cybernetics Inc., Rockville, MD, USA).
6
Colon Tissue Histochemistry and Immunostaining
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The mouse colon tissue was fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and cut into sections at a thickness of 4 µm. For immunohistochemical staining, the colonic sections were subjected to deparaffinization, hydration, antigen retrieval, quenching of endogenous peroxidase, and blocking procedures. All slices were then incubated with the primary antibodies against pSTAT3 and Ki-67 (Figure S2
7
Western Blot Analysis of Apoptosis Markers
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Caspase-3, Bax and cytochrome c were detected by western blot assay in rat hippocampal homogenates. Protein samples were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis. The separated proteins were then transferred from the gel onto a nitrocellulose membrane. The membranes were blocked in 5% fat-free milk prepared in phosphate-buffered saline/Tween-20 (PBST) buffer for 1 hour, and incubated with rabbit primary antibodies against activated caspase-3, Bax and cytochrome c (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C. The membranes were washed three times with PBST, and then incubated with goat anti-rabbit IgG (1:20,000; ZSGB-BIO, Beijing, China) for 2 hours at room temperature. Blots were developed using a 3,3′-diaminobenzidine kit (ZSGB-BIO). Semiquantitative analysis of the blots was performed with a FM 0442 gel imaging system (ProteinSimple, Hercules, CA, USA). Each experiment was performed in triplicate. The final optical density value was the average of the three separate analyses. Protein levels were expressed as the optical density ratio of the target protein to β-actin (reference).
8
Immunohistochemical Analysis of AFP and CXCR4 in HCC
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The expression and cellular distribution of AFP and CXCR4 proteins in HCC specimens were assessed by immunohistochemical analysis. Five‐millimetre‐thick paraffin sections were deparaffinized and rehydrated according to standard protocols, and heat‐induced antigen retrieval was performed in sodium citrate buffer (10 mmol/l, pH 6.0). Endogenous peroxidase was inhibited by 0.3% H2O2, and non‐specific protein binding was blocked with 10% goat serum. The sections were then incubated with primary antibody against AFP and CXCR4 (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. Non‐immune immunoglobulin G was used as a negative control, and antigenic sites were localized using a SP9000 Polymer Detection System and a 3,3′‐diaminobenzidine kit (ZSGB‐BIO, Beijing, China).
9
Immunohistochemical Evaluation of Tumor Markers
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The protein expression of c-myc, Ras, activated caspase-3 and PARP-1 in tumor-bearing mice were evaluated by immunohistochemical analysis. Briefly, tumorous tissue was removed from tumor-bearing mice and cut into 5-mm-thick paraffin sections. The sections were deparaffinized and rehydrated according to standard protocols. The sections were then incubated with primary antibody (1:100 dilution; Abcam Trading Company, Ltd. Shanghai, China) at 4 °C overnight. Nonimmune (IgG) was used as a negative control, and antigenic sites were identified using an SP9000 Polymer Detection System and a 3,3’-diaminobenzidine kit (ZSGB-BIO, Beijing, China). The standard protocols were performed in accordance with approved guidelines [19 (link), 20 (link)].
10
Immunohistochemical Analysis of HCC Markers
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The expression and cellular distribution of AFP, Ras and CXCR4 proteins in HCC specimens were assessed by immunohistochemical analysis. Five-millimeter-thick paraffin sections were deparaffinized and re-hydrated according to standard protocols, and heat-induced antigen retrieval was performed in sodium citrate buffer (10 mmol/L, pH 6.0). Endogenous peroxidase was inhibited by 0.3% H2O2, and non-specific protein binding was blocked with 10% goat serum. The sections were then incubated with primary antibody against AFP, Ras and CXCR4 (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. Non-immune immunoglobulin G(IgG) was used as a negative control, and antigenic sites were localized using a SP9000 Polymer Detection System and a 3,3′-diaminobenzidine kit (ZSGB-BIO, Beijing, China).
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