Immunohistochemistry was performed by the Envision two-step method [cat. no. ZM-0166 (Beijing Zhongshang Jinqiao Co.); K5007 (Dako)]. The primary antibody was raised against Ki67 (cat. no. ZM-0166, 1:200 dilution) and K5007 (Dako; no dilution) was used as the secondary antibody. The steps included: i) Dewaxing with hot water; ii) antigen repair under high pressure citric with acid at pH 6.0; iii) hydrogen peroxide blocking of endogenous peroxidase; iv) primary antibody incubation at 37°C for 30 min; v) secondary antibody incubation at 37°C for 15 min; vi) DAB staining at 22°C for 5 min; vii) dehydration and mounting. We compared conventional hematoxylin and eosin (H&E) staining with Ki67 DBA immunostaining and defined + as >0 and ≤25%; ++ as >25 and ≤50%; +++ as >50 and ≤75%; and ++++ as >75% (Fig. 2 ).
K5007
Manufactured by Agilent Technologies
Sourced in Denmark, United States, United Kingdom, China, Germany, Australia
The K5007 is a high-performance power supply unit designed for laboratory and industrial applications. It features a wide input voltage range and provides stable and precise output voltages and currents. The core function of the K5007 is to supply power to various electronic devices and systems under test in a controlled and regulated manner.
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Lab products found in correlation
S2369, by Agilent Technologies (3 mentions)
Ab205921, by Abcam (2 mentions)
S2023, by Agilent Technologies (2 mentions)
K3468, by Agilent Technologies (2 mentions)
S2367, by Agilent Technologies (2 mentions)
Eclipse ti sr, by Nikon (2 mentions)
Histovt one, by Nacalai Tesque (2 mentions)
Gb23303, by Wuhan Servicebio Technology (2 mentions)
Dfc295, by Leica (2 mentions)
Cell death detection kit, by Roche (2 mentions)
Silver staining kit, by Thermo Fisher Scientific (2 mentions)
Bc3710, by Solarbio (2 mentions)
Chemidoc touch, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Fujifilm (2 mentions)
Dako real envision detection system k5007, by Agilent Technologies (1 mentions)
Dako real envision detection system, by Agilent Technologies (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Ab181606, by Abcam (1 mentions)
G1005, by Wuhan Servicebio Technology (1 mentions)
77 protocols using k5007
1
Immunohistochemical Evaluation of Ki67 Expression
2
Immunohistochemical Analysis of Myeloperoxidase and p65
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The endogenous peroxidases in 4 μm-thick slices were deactivated by incubation with 3% H2O2 for 10 min. For antigen retrieval, the sections were soaked in 10 mM citrate buffer solution (pH 6.0) and heated twice in a microwave oven. After washed thoroughly with PBS (pH7.4), the sections were blocked with 3% BSA in tris buffered saline (TBS) for 20 min, then incubated with anti-myeloperoxidase antibody (anti-MPO) (1:200, #SH0022, Skyhobio) and anti-p65 (1:400, #SH0023, Skyhobio) antibodies overnight at 4°C followed by incubation with HRP-conjugated secondary antibody (#K5007, Dako) for 50 min. The sections were further incubated with DAB-H2O2 solution (#K5007, Dako), counterstained with hematoxylin, dehydrated with ethanol and sealed in resinene for microscopic observation.
3
Evaluating [99mTc]Tc-HYNIC-PTP for Tumor Imaging
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The feasibility of [99mTc]Tc-HYNIC-PTP as a SPECT probe for tumor imaging was examined using established subcutaneous tumor models. Briefly, [99mTc]Tc-HYNIC-PTP solution (200 µL, [99mTc]Tc = 10 mCi/mL, corresponding to 2 µg of HYNIC-PTP) was intravenously injected into tumor-bearing mice. SPECT images were acquired 0.5, 1, 2, and 4 h after injection using a SPECT imaging system equipped with a Xeleris 2.0 workstation and low-energy general-purpose collimators (Infinia, Denver, CO, USA). After SPECT imaging, the tumors and muscle tissues were excised to confirm plectin expression levels by immunohistochemistry. Briefly, excised specimens were fixed in formalin and embedded in paraffin. The sections (5 μm) were subjected to heat treatment in a citrate solution for antigen retrieval and blocking with 3% bovine serum albumin for 30 min. Then the sections were incubated with anti-plectin primary antibody (1:200) overnight at 4 °C. After incubation with secondary antibody (K5007, DAKO, Carpinteria, CA, USA) at room temperature for 50 min, the slides were treated with 1:100 DAB (K5007, DAKO, USA) and counterstained with hematoxylin. At least three sections of each specimen were chosen, and 5–10 high-power visual fields were randomly selected to calculate the positive area of staining. The average areas were then used to plot histograms.
4
Immunohistological Analysis of Fabricated Cell Sheets
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Immunohistology was performed according to methods described in our previous article30 (link). Fabricated cell sheets were fixed in 4% paraformaldehyde (Wako Pure Chemical Industries), dehydrated, embedded in paraffin, sectioned (4 μm) and mounted on glass slides. The slides were de-paraffinized, and antigen retrieval was performed by autoclaving with citrate buffer (Histo-VT One, Nacalai Tesque). Peroxidase blocking (Dako) and protein blocking (Nacalai Tesque) were carried out to prevent non-specific reactions. The slides were then incubated overnight at 4 °C in PBS containing primary antibody (Table S2 ). After washing with PBS, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse, HRP; K5007; Dako) at room temperature for 1 h. The sections were washed again and treated with 3,3′-diaminobenzidine (K5007; Dako) to visualize the HRP. Nuclei were stained by hematoxylin. De-paraffinized sections were also stained with hematoxylin and eosin (HE) for histological evaluation.
5
Immunohistochemical Analysis of Collagen
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The paraffin-embedded samples were sectioned, dewaxed to water, and underwent antigen extraction. After incubating the samples at room temperature for 30 min with 3% hydrogen peroxide in the dark, 10% goat serum (K5007, Dako, Shanghai, China) was added and the samples were maintained at 25 °C for 1 h. The samples were then incubated overnight at 4 °C with antibodies to COLL-I (14695-1-AP, San Ying Biotechnology, Wuhan, China), COLL-II (MA1-37493, Thermo Fisher Scientific, USA), and COLL-III (22734-1-AP, San Ying Biotechnology) at appropriate dilutions. On the second day, the samples were added to a 37 °C constant temperature incubator with enhanced chemical reaction solution for 20 min and washed with PBS before goat anti-rabbit secondary antibody (K5007, Dako) was added to cover the labeled tissue. The samples were incubated at room temperature for 50 min. The color was developed by mixing with diaminobenzidine solution (DA1016, Solarbio) at room temperature for 5–10 min before being washed with distilled water, re-stained, dehydrated, and sealed. Positive fluorescence microscopy (NIKON ECLIPSE E100, Nikon) and panoramic scanning (NIKON DS-U3, Nikon) were performed using an imaging system.
6
Immunohistochemical Analysis of NF-κB Expression
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After deparaffinization and rehydration of the slides, the tissue sections were treated with 3% H2O2 (in methanol) for 10 min. After washing with distilled water for 2 min (3X), the slides were placed in citrate buffer (0.01 M, pH = 6) and heated to boiling for 10 min. The slides were allowed to cool at RT. 2% bovine serum albumin mixed in normal sheep serum (Avicenna Research Institute, Tehran, Iran) was used as blocking agent. After that, the slides were incubated with a primary antibody for NF-κB, (ab16502, Abcam, 1:500) overnight at 4 °C. After washing, slides were incubated with secondary antibody (K5007, DAKO) for 1 h at RT. To visualize immunoreactivity, 3, 3’-Diaminobenzidine (DAB) made up with substrate buffer (K5007, DAKO), was added to slides. Finally, the sections were counterstained with Mayer’s haematoxylin (Sigma) for 2 min, dehydrate, and mounted. The percent of NF-κB expression was quantitatively analysed by ImageJ software (ImageJ, NIH, Bethesda, MD, USA). The slides were examined using a microscope (Olympus BX51) connected to a digital camera (Olympus, DP71) by a veterinary anatomic pathologist.
7
Immunohistochemical Analysis of Immune Cells
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For anti-CD4, CD8, CD45RO (UCHL-1) and CD68, the cell count was performed using five observed fields. Briefly, four lm-thick sections were dewaxed and rehydrated in graded ethanol. The primary antibodies used were CD4 (IS 649; Dako) RTU, CD8 (IS623; Dako) RTU, CD45RO (AM113-5M; Biogenex) RTU, and CD68 (IS 613, Dako) RTU.
For all antibodies, a steamer was used for epitope retrieval with either citrate buffer or Tris-EDTA buffer. The Advance polymer (K 5007; Dako) was used as a reaction amplifier. Visualization of the antibody complex was achieved using 3.3-diaminobenzidine tetrahydrochloride (K 5007; Dako) according to the manufacturer's instructions. Sections were counterstained with Harris’ hematoxylin.
Appropriate negative and positive controls were included in each assay. The expression of these markers was calculated as the percentage of positivity in relation to the total amount of nuclei counted per se ction.
For all antibodies, a steamer was used for epitope retrieval with either citrate buffer or Tris-EDTA buffer. The Advance polymer (K 5007; Dako) was used as a reaction amplifier. Visualization of the antibody complex was achieved using 3.3-diaminobenzidine tetrahydrochloride (K 5007; Dako) according to the manufacturer's instructions. Sections were counterstained with Harris’ hematoxylin.
Appropriate negative and positive controls were included in each assay. The expression of these markers was calculated as the percentage of positivity in relation to the total amount of nuclei counted per se ction.
8
Immunohistochemical Analysis of Colorectal Samples
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Paraffin-embedded TMA samples were cut into 4 μm sections. Rabbit antibodies for PD-L1 (SP142, spring bioscience), PD-1 (SP269, spring bioscience), CD4 (ZA-0519, ZSGB-BIO), CD8 (ZA-0508, ZSGB-BIO), MLH1 (ZM-0154, ZSGB-BIO), MSH2 (ZA-0622, ZSGB-BIO), MSH6 (ZA-0541, ZSGB-BIO) and PMS2 (ZA-0542, ZSGB-BIO) were used. All the primary antibodies we used were also clinically applied in pathology department of our hospital. IHC assays were performed as follows. Briefly, the tissue sections were baked, deparaffinized and rehydrated. Endogenous peroxide was blocked by incubating with 3% hydrogen peroxide in methanol for 15 minutes at 37°C. Then processed for antigen retrieval by high pressure cooking in EDTA antigen retrieval solution (pH=8) for about 10 minutes. Sections were incubated with primary antibodies at 37°C for about 1.5h. After washing, tissue sections were treated with HRP RABBIT/MOUSE secondary antibody (K5007, 20029103, Dako) for 30 minutes at room temperature. The sections were immunostained with diaminobenzidine tetrahydrochloride (DAB, K5007, 20019193, Dako) and counterstained with hematoxylin. Immune cells in the lymphoid tissue within normal colorectal tissue cores served as positive controls for the staining of PD-L1, PD-1, CD4 and CD8.
9
Immunohistochemical Staining Protocol
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Tissue samples were paraffin-embedded and cut into 5-µm sections. The slides were then heated at 65 °C for 120 min, followed by de-paraffinized, hydrated and antigen retrieval. Endogenous peroxidases were blocked with 3% peroxide for 15 min, then in 5% BSA for 1 h and incubated overnight at 4 °C with the indicated primary antibodies. Subsequently, anti-rabbit/mouse secondary antibodies (K5007, Dako) were applied and incubated for 60 min at 37 °C. Signals were revealed using freshly prepared DAB substrate solution (K5007, Dako) at room temperature for 5 min. Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. Staining results were captured using microscopy (DM4000B, Leica). Each section was evaluated by two independent pathologists blinded to the clinical status of patients and graded according to the positive staining intensity scores (no staining, 0; weak staining, 1; moderate staining, 2; strong staining, 3) and the expression extent scores (< 25%, 1; 25–50%, 2; 50–75%, 3; > 75%, 4).
10
Immunohistochemical Analysis of EndMT Drivers
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The immunohistochemistry method was used for all EndMT drivers. Briefly, lung tissue sections were deparaffinised in xylene and ethanol, respectively, followed by antigen retrieval performed at 110 °C for 15 min using target retrieval citrate buffer pH 6.0 (Dako S2369, Mulgrave, VIC, Australia) in a decloaking chamber (Biocare Medical, Pacheco, CA, USA). Primary antibodies were used to stain tissues for 60 min: mouse monoclonal TGF-β1 (1:2000, Abcam ab27969, Melbourne, VIC, Australia), polyclonal phospho-Smad2/Smad3 (pSmad-2/3, 1:100, ThermoFisher PA5-110155, Scoresby, VIC, Australia), mouse monoclonal Smad-7 (1:50, Santa Cruz Biotechnology sc-101152, Dallas, TX, USA), and rabbit monoclonal anti-β-catenin (1:100, Abcam ab32572, Melbourne, VIC, Australia), followed by secondary HRP rabbit/mouse antibodies (Dako K5007, Mulgrave, VIC, Australia). The DAB substrate (Dako K5007, Mulgrave, VIC, Australia) was added to make the protein markers visible, and hematoxylin (Australian Biostain P/L, Traralgon, Australia) was used to counterstain the nucleus. We have previously published using these techniques [4 (link),11 (link),12 (link),13 (link),14 (link),15 (link),16 (link),17 (link)].
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