TSA plus fluorescent multiple staining kit (#G1236-100T, Servicebio) was applied for multiplex IHC. The 5-mm formalin-fixed and parrffin-embedded slides were deparaffinized and rehydrated. All slides were subjected to epitope retrieval with EDTA (pH 8.0) for 8 min. After cooling (25℃), slides were washed with PBS (3*5 min), and endogenous peroxidase activity were blocked with H2O2 (3%) for 25 min. Then blocking buffer (5%BSA, Solarbio, #SW3015) was used for 30 min-protein blocking. All slides were incubated with antibody against CD8 (#GB13068, Servicebio, 1:500) at 4℃ overnight. After washes, sections were incubated with an HRP-conjugated secondary antibody for 50 min. Then slides were dyed with CY3-TSA for 10 min. This method was applied three more times using the antibodies as follows, GINS2 (Proteintech, #16247-1-AP, 1:1000, dye FITC), CD4 (Wisee Biotechnology, #YX32005-100, 1:1000, dye 647-TSA), and CD3 (Servicebio, #GB13440, 1:100, dye594). EDTA (pH 8.0) buffer was applied for the next round of epitope retrieval with a cooker (125℃, 15 min). Cell nucleus was labeled with DAPI (Servicebio, #G1012) and covered with Antifade Mounting Medium (Beyotime, #P0126). Secondary antibodies were used as follows: anti-rabbit (1:500, Servicebio, GB23303) and anti-mouse (1:500, Servicebio, GB23301).
Gb13440
Manufactured by Wuhan Servicebio Technology
Sourced in United States
GB13440 is a laboratory equipment product. It is a general-purpose device used for various applications in scientific research and laboratory settings. The core function of GB13440 is to perform specific tasks required in the laboratory environment. However, a detailed description of its functionality cannot be provided while maintaining an unbiased and factual approach. Further information may be available from the manufacturer or through additional research.
Automatically generated - may contain errors
Lab products found in correlation
Ab214488, by Wuhan Servicebio Technology (2 mentions)
Pannoramic midi, by 3DHISTECH (2 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Sw3015, by Solarbio (1 mentions)
Gb13068, by Wuhan Servicebio Technology (1 mentions)
Gb23303, by Wuhan Servicebio Technology (1 mentions)
Gb23301, by Wuhan Servicebio Technology (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Gb21301, by Wuhan Servicebio Technology (1 mentions)
Gb22303, by Wuhan Servicebio Technology (1 mentions)
G1012, by Wuhan Servicebio Technology (1 mentions)
Gb113885, by Wuhan Servicebio Technology (1 mentions)
Gb13340, by Wuhan Servicebio Technology (1 mentions)
Gb12068, by Wuhan Servicebio Technology (1 mentions)
Gb113150, by Wuhan Servicebio Technology (1 mentions)
Cd163, by Wuhan Servicebio Technology (1 mentions)
Caseviewer, by 3DHISTECH (1 mentions)
5 protocols using gb13440
1
Multiplex IHC with Fluorescent Staining
2
Quantifying Tumor-Infiltrating T Cells
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Tumor cells were seeded at 3×104 cells per well in glass slides and cultured for 12 hours. Then activated T cells were added to the culture in the absence or presence of F7AK3 (1 µg/mL) for 30 min. Afterwards, cells were washed twice with PBS and fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Cells were incubated with anti-TROP2 (Abcam, ab214488) and anti-CD3 (Servicebio, GB13440) overnight at 4°C. Secondary fluorescent antibodies conjugated with FITC and Cy3 (Servicebio, GB22303 and GB21301) were added for 1 hour and 4',6-diamidino-2-phenylindole (DAPI) (Servicebio, G1012) was used for nuclear staining. The percentages of CD3+ T cells in tumor tissues were quantified manually as the percentage of green labeled cells (CD3+ T cells) to all cells (DAPI). Images were obtained with a confocal microscope (Olympus) under a 60×oil objective.
3
Immunohistochemical Analysis of TROP2 and CD3
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Paired tumor and adjacent non-tumor tissue samples were obtained with informed consent from Tongji Hospital of HUST (Wuhan, China). The demographic and clinical characteristics of the enrolled patients are presented in online supplemental table S1 . The specimens were isolated at the time of surgery, formalin fixed and paraffin embedded, and stained with H&E. TROP2 expression was scored as follows: multiplication of the intensity of immunostaining ((1) mild; (2) moderate; (3) strongly positive) and the percentage of positive tumor cells, which resulted in a score of 0–300. A score <10 was considered as 0, a score of 10–40 was considered as 1, 41–140 as 2 and 141–300 as 3. Standard immunohistochemistry was performed with antibodies against TROP2 (Abcam, ab214488), CD3 (Servicebio, GB13440) then examined by two blinded pathologists.
4
Multiplexed Immunofluorescence Staining of Colorectal Tumors
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Multiplex immunofluorescence staining was performed at Servicebio Technology Co., Ltd. (Wuhan, China), and a multiplexed tyramide signal amplification method was used on 4 μm sections of the tissue microarray (TMA) containing 30 pairs of colorectal tumors and adjacent normal tissues from the Nanjing cohort. The details of TMA were reported previously[22 (link)]. After deparaffinization, antigen retrieval, and serum blocking, the TMA was sequentially stained with the primary antibodies anti-CD3 (GB13440, Servicebio), anti-CD45 (GB113885, Servicebio), anti-CD22 (66103-1-IG, Proteintech, Rosemont, IL, USA) and anti-FEN1 (14768-1-AP, Proteintech), followed by the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) and tyramine signal amplification (TSA) visualization. DAPI (4′,6-diamidino-2-phenylindole) was finally added to stain nuclei until all four markers were labeled. Antibody details are shown in Supplementary Table 5 (available online). The slide was scanned with the Pannoramic MIDI (3DHISTECH, Hungary). Multispectral images were evaluated using the HALO Image Analysis Platform (Version 3.0.311.314, Indica, US). Basophils (CD22+), eosinophils (CD45+), T cells (CD3+) and FEN1-positive cells were quantified, and their average intensity in TMA was calculated.
5
Multiplexed Immunofluorescence Analysis of Kidney Biopsies
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All LN biopsy samples and normal renal control tissues were processed into paraffin blocks, then, the paraffin embedded samples were sliced into 4-µm sections. After deparaffinization and rehydration, the tissues were subjected to hematoxylin and eosin (HE) as well as multiplexed immunofluorescence staining. The detailed procedure has been described elsewhere [18 (link)]. Five immune cell classes were probed: B cells (CD19: GB11061, Servicebio), T cells (CD3, GB13440, Servicebio), CD8 + T cells (CD8, GB12068, Servicebio), macrophages (CD68, GB113150, Servicebio), and CD163 + macrophages (CD163, GB13340, Servicebio). The slides were scanned using an automatic digital slide scanner Pannoramic MIDI (3DHISTECH, Hungary) and analyzed using CaseViewer (3DHISTECH, Hungary).
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