Tissue microarray of TNBC patients with information of clinical–pathological parameters was purchased from Outdo Biotech (HBreD090Bc01; Shanghai, China). Paraffin‐embedded sections of xenograft tissues were subjected to deparaffinization and rehydration. H/E staining of sections was carried out using H/E staining kit (Beyotime, Shanghai, China) according to manufacturer’s instructions. For immunohistochemical staining of tissue microarray and sections of xenograft and antigen retrieval, blocking of non‐specific binding and incubation of primary antibodies at 4 °C overnight was sequentially conducted. The primary antibodies used were list as follows: anti‐phospho‐EGFR (ab40815; Abcam, 1 : 200) and anti‐SGLT1 (ab14686; Abcam, 1 : 100). After incubation with secondary goat anti‐rabbit immunoglobulin conjugated to peroxidase‐labelled dextran polymer (SV0002; Boster) at 37 °C for 1 h, visualization, counterstaining with haematoxylin and mounting were performed. Semiquantitative evaluations of protein expression were scored on the basis of the intensity and the percentage of phospho‐EGFR‐ or SGLT1‐positive tumour cells as previously described (Wang et al., 2014 ).
Sv0002
Manufactured by Boster Bio
Sourced in United States, China
The SV0002 is a laboratory centrifuge designed for general-purpose applications. It features a compact and durable construction, with a maximum speed of 6,000 RPM and a capacity of up to 6 x 50 mL tubes. The centrifuge is suitable for a variety of sample preparation and separation tasks, including but not limited to, pelleting cells, clarifying solutions, and precipitating macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Hbred090bc01, by Shanghai Outdo Biotech Co. (3 mentions)
Hpa001122, by Merck Group (2 mentions)
Sv0001, by Boster Bio (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab40815, by Abcam (1 mentions)
Ab14686, by Abcam (1 mentions)
He staining kit, by Beyotime (1 mentions)
Ab126625, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab23446, by Abcam (1 mentions)
Ab34712, by Abcam (1 mentions)
Bs 0032r, by Bioss Antibodies (1 mentions)
Ab38898, by Abcam (1 mentions)
Bs 20658r, by Bioss Antibodies (1 mentions)
Ab28364, by Abcam (1 mentions)
Ba0407, by Boster Bio (1 mentions)
3 3 diaminobenzidine (dab), by ZSGB-BIO (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
9 protocols using sv0002
1
Tissue Microarray Analysis of TNBC Patients
2
Tissue Microarray Analysis of TNBC
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Tissue microarray of TNBC patients with information of clinic-pathological parameters was purchased from Outdo Biotech (HBreD090Bc01; Shanghai, China). Tissue samples were pre-stained with Ki67. All procedures were approved by the Ethical Committee of Tongji Hospital, China. Informed consent was obtained from all subjects. For immunohistochemical staining, antigen retrieval, blocking of non-specific binding and incubation of primary antibodies at 4 °C overnight were conducted sequentially. The primary antibody of anti-WDHD1 (HPA001122, Sigma-Aldrich, 1:500) was used. After incubation with secondary goat anti-rabbit immunoglobulin conjugated to peroxidase-labelled dextran polymer (SV0002; Boster) at 37 °C for 1 h, visualisation, counterstaining with haematoxylin and mounting were performed. Semi-quantitative evaluations of protein expression were scored on the basis of the intensity and the percentage of WDHD1-positive tumour cells as previously described59 (link)–62 (link).
3
Immunohistochemical Analysis of CHAF1A and Ki-67
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Immunohistochemistry (IHC) was performed on paraformaldehyde-fixed paraffin sections. The following antibodies were used in IHC along with a streptavidin peroxidase conjugate: rabbit CHAF1A (ab126625, 1:100; Abcam) and Ki-67 (ab15580, 1:200; Abcam) antibodies, and a rabbit secondary antibody kit (SV0002; Boster Biotechnology) was applied. The IHC steps were performed as reported earlier.23 (link) After all steps were completed, IHC staining was scored independently in a blinded situation by two experienced pathologists. A semiquantitative scoring system based on the product of staining intensity and together with the percentage of positive liver cells was applied in this study. Immunostaining intensity was evaluated as the following four grades: 0, negative; 1, weak; 2, moderate; 3, strong, and the percentage of positive cells was categorized into the following groups: 0, 0%; 1, 1%–10%; 2, 11%–50%; 3, 51%–80%; and 4, >80%. The immunostaining intensity and average percentage of positive cells were evaluated for ten high independent magnification fields. Then, the final weighed expression score was obtained (0–12) after multiplying the staining intensity by the percentage of positive cells.
4
Immunohistochemical Profiling of Articular Cartilage
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The method of sampling is the same as Safranin O-Fast green staining. Articular cartilage tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The block was cut into 6 μm sections, and slides were prepared. Slides were incubated in primary antibodies for MMP8 (BA2201, dilution 1:100, Boster), MMP9 (ab38898, dilution 1:100, Abcam), COL1A1 (ab23446, dilution 1:100, Abcam), COL2A1 (ab34712, dilution 1:100, Abcam), and BCL2 (bs-0032R, dilution 1:200, Bioss) at 4 °C overnight. The next day, slides were washed three times and incubated with polyclonal antibodies against rabbit IgG-HRP (#SV0002, Boster) and mouse IgG-HRP (#SV0001, Boster) at room temperature for 1 h. Slides were then stained with 1 μM DAPI (Thermo Fisher Scientific, Waltham, MA, USA) to visualize cell nuclei.
5
Immunohistochemical Detection of FSHR
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Cells were fixed in 4% paraformaldehyde for 15 min and washed with PBS three times for 3 min each time. Cells were then incubated in a 3% hydrogen peroxide solution (H2O2) at room temperature for 10 min to inactivate endogenous peroxidase and washed with PBS three times for 5 min each time. Subsequently, 5% BSA was added to the petri dish and cells were blocked at room temperature for 30 min. The primary antibody rabbit polyclonal FSHR (1:500, bs-20658R, Bioss) was added dropwise and incubated in wet box overnight at 4 °C. Goat anti-rabbit IgG secondary antibody marked by horseradish peroxidase (HRP) (1:500, SV0002, Boster) was added dropwise, and the cells were incubated at room temperature for 30 min. Diaminobenzidine tetrahydrochloride (DAB) was added as a substrate and stained for 5–10 min. Next, cells were flushed fully with running water and counterstained with hematoxylin for 3 min. Finally, cells were conventionally dehydrated with an ethanol and xylene gradient and mounted in neutral resin. Immunoreactivity was detected using a fluorescence microscope.
6
Immunohistochemical Analysis of WDHD1 in TNBC
7
Immunohistochemical Analysis of Angiogenesis
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Tissue sections (from 5 mice in each group) were incubated with 3% H2O2 for 5 min, boiled in 0.01 M citrate buffer solution (pH 6.0) for 10 min, blocked with 5% bovine serum albumin for 1 h at room temperature (RT), and incubated with primary antibodies (anti‐CD31, ab28364, 1:50; Abcam; anti‐VEGFA, BA0407, 1:200; Boster, CDK6, MA5‐13333, 1:100; Invitrogen) at 4°C overnight. The sections were then incubated with the corresponding horseradish peroxidase (HRP)‐conjugated secondary antibodies (anti‐mouse, SV0001; Boster; anti‐rabbit, SV0002; 1:10,000; Boster) at RT for 1 h, followed by DAB (ZSGB‐BIO) and hematoxylin (Guangzhou Xiuwei Company) staining. Images (3 images /section) were captured under a microscope (NIKON ECLIPSE CI). The area of positive staining was measured using Image‐Pro Plus 6.0 software (Media Cybernetics Corporation).
8
Paraffin Tissue Sectioning and Immunohistochemistry
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After the fixed tumor tissue was embedded in paraffin, tissue was sectioned into very thin (5–10 μm) sections using a microtome, and these paraffin sections were placed on slides and kept warm in an oven at 60 °C for 2 h. Then, the sections were deparaffinized with xylene for 15 min, and the tissue was successively rehydrated with 100%, 90%, 80%, and 70% alcohol for 5 min and stained with hematoxylin and eosin for 2–3 min (C01055, Beyotime, Shanghai, China), and the tissue was then dehydrated successively with 80%, 90%, and 100% alcohol and finally with xylene for 5 min. Finally, the neutral resins were mounted and left to dry overnight to create permanent slides. The primary antibodies rabbit anti-Ki 67 antibody (1:200, 27309-1-AP, Proteintech, WuHan, China) and rabbit anti-Caspase 3 antibody (1:200, 19677-1-AP, Proteintech, WuHan, China) were incubated overnight according to the instructions of the immunohistochemistry kit (SV0002, BOSTER, Wuhan, China) used in this study. The next day, the secondary antibody was incubated for 30 min and then DAB stained and observed under the microscope so that pictures could be taken.
9
Histological Analysis of Femoral Heads
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Prior to the assay, all femoral head samples were fixed in 4% paraformaldehyde solution at room temperature for 72 h and subsequently subjected to decalcification in 0.5 M EDTA for 16 weeks prior to dehydration, paraffin embedding and serial sectioning (mouse femoral samples were fixed in 4% paraformaldehyde for 12 h at 4 °C and decalcified for 4 weeks). The samples were cut into paraffin sections (4 μm) for subsequent hematoxylin and eosin staining with a commercial staining kit (DM0064, Boster Biological Technology, Wuhan, China) according to the manufacturer’s instructions. For immunofluorescence (IF) staining, prepared sections were incubated with anti-ACKR1 and anti-vWF antibodies. FITC- or Cy3-conjugated secondary antibodies were used to display the signals. For immunohistochemistry (IHC), a commercial HRP-DAB system kit (SV0002, Boster) was used to display the signal of the primary antibodies. Information on all antibodies is shown in Supplementary Table 3 .
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